Crystalline Peptide Epoxy Ketone Protease Inhibitors and the Synthesis of Amino Acid Keto-Epoxides

ABSTRACT

The invention relates to crystalline peptide keto epoxide compounds, methods of their preparation, and related pharmaceutical compositions. This invention also relates to methods for the preparation of amino acid keto-epoxides. Specifically, allylic ketones are stereoselectively converted to the desired keto epoxides.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/287,043, filed Oct. 3, 2008, which claims the benefit of U.S.Provisional Application Ser. No. 60/997,613, filed Oct. 4, 2007, andU.S. Provisional Application Ser. No. 61/008,987, filed Dec. 20, 2007,each of these earlier filed applications is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I antigenpresentation, apoptosis, cell growth regulation, NF-κB activation,antigen processing, and transduction of pro-inflammatory signals.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits organized into four rings. Inyeast and other eukaryotes, 7 different α subunits form the outer ringsand 7 different β subunits comprise the inner rings. The α subunitsserve as binding sites for the 19S (PA700) and 11S (PA28) regulatorycomplexes, as well as a physical barrier for the inner proteolyticchamber formed by the two β subunit rings. Thus, in vivo, the proteasomeis believed to exist as a 26S particle (“the 26S proteasome”). In vivoexperiments have shown that inhibition of the 20S form of the proteasomecan be readily correlated to inhibition of 26S proteasome. Cleavage ofamino-terminal prosequences of β subunits during particle formationexpose amino-terminal threonine residues, which serve as the catalyticnucleophiles. The subunits responsible for catalytic activity inproteasomes thus possess an amino terminal nucleophilic residue, andthese subunits belong to the family of N-terminal nucleophile (Ntn)hydrolases (where the nucleophilic N-terminal residue is, for example,Cys, Ser, Thr, and other nucleophilic moieties). This family includes,for example, penicillin G acylase (PGA), penicillin V acylase (PVA),glutamine PRPP amidotransferase (GAT), and bacterialglycosylasparaginase. In addition to the ubiquitously expressed βsubunits, higher vertebrates also possess three interferon-γ-inducible βsubunits (LMP7, LMP2 and MECL1), which replace their normalcounterparts, X, Y and Z respectively, thus altering the catalyticactivities of the proteasome. Through the use of different peptidesubstrates, three major proteolytic activities have been defined for theeukaryote 20S proteasome: chymotrypsin-like activity (CT-L), whichcleaves after large hydrophobic residues; trypsin-like activity (T-L),which cleaves after basic residues; and peptidylglutamyl peptidehydrolyzing activity (PGPH), which cleaves after acidic residues. Twoadditional less characterized activities have also been ascribed to theproteasome: BrAAP activity, which cleaves after branched-chain aminoacids; and SNAAP activity, which cleaves after small neutral aminoacids. The major proteasome proteolytic activities appear to becontributed by different catalytic sites, since inhibitors, pointmutations in β subunits and the exchange of γ interferon-inducing βsubunits alter these activities to various degrees.

What is needed are improved compositions and methods for preparing andformulating proteasome inhibitor(s).

SUMMARY OF THE INVENTION

The invention generally relates to the synthesis of proteasomeinhibitors and the preparation and purification of intermediates usefultherefor.

One aspect of the invention relates to crystalline compounds having astructure of Formula (I) or a pharmaceutically acceptable salt thereof,

wherein

X is O, NH, or N-alkyl, preferably O;

Y is NH, N-alkyl, O, or C(R⁹)₂, preferably N-alkyl, O, or C(R⁹)₂;

Z is O or C(R⁹)₂, preferably C(R⁹)₂;

R¹, R², R³, and R⁴ are hydrogen;

each of R⁵, R⁶, R⁷, R⁸, and R⁹ is independently selected from hydrogen,C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl,each of which is optionally substituted with a group selected fromalkyl, amide, amine, carboxylic acid or a pharmaceutically acceptablesalt thereof, carboxyl ester, thiol, and thioether, preferably R⁵, R⁶,R⁷, and R⁸ are independently selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl,and C₁₋₆aralkyl and each R⁹ is hydrogen, more preferably, R⁶ and R⁸ areindependently C₁₋₆alkyl, R⁵ and R⁷ are independently C₁₋₆aralkyl andeach R⁹ is H;

m is an integer from 0 to 2; and

n is an integer from 0 to 2, preferably 0 or 1.

Another aspect of the invention relates to a crystalline compound ofFormula (III)

wherein X is any suitable counterion.

Another aspect of this invention relates to methods for the synthesis ofamino acid keto-epoxides according to scheme (I)

wherein

-   R¹ is selected from a protecting group or a further chain of amino    acids, which itself may be optionally substituted, preferably a    protecting group, most preferably an electron withdrawing protecting    group;-   R² is selected from hydrogen and C₁₋₆alkyl;-   R³ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkoxyalkyl,    heterocyclyl, aryl, heteroaryl, C₁₋₆heteroaralkyl, and C₁₋₆aralkyl;    and-   wherein the method comprises a stereoselective epoxidation under    epoxidizing conditions, preferably an aqueous sodium hypochlorite    (bleach) or calcium hypochlorite solution in the presence of a    cosolvent selected from pyridine, acetonitrile, dimethylformamide    (DMF), dimethylsulfoxide (DMSO), N-methylpyrrolidine (NMP),    dimethylacetamide (DMA), tetrahydrofuran (THF), and nitromethane.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a DSC (differential scanning calorimetry) thermogram ofcrystalline compound 1.

FIG. 2 shows an XRPD (X-ray powder diffraction) pattern of crystallinecompound 1.

FIG. 3 shows a TG thermogram of crystalline compound 1.

FIG. 4 shows a DSC thermogram of amorphous compound 1 compared to a DSCthermogram of crystalline compound 1.

FIG. 5 shows an XRPD pattern of amorphous compound 1 compared to theXRPD pattern of crystalline compound 1.

FIG. 6 shows a TG thermogram of amorphous compound 1 compared to the TGpattern of crystalline compound 1.

FIG. 7 shows a DSC curve of an amorphous sample of compound 1.

FIG. 8 shows the XRPD pattern of amorphous compound 1.

FIG. 9 shows a DSC curve of a crystalline compound F.

FIG. 10 shows an XRPD pattern of a crystalline compound F.

FIG. 11 shows a DSC curve of a crystalline citrate salt of compound 1.

FIG. 12 shows an XRPD pattern of a crystalline citrate salt of compound1.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the invention relates to crystalline compoundshaving a structure of Formula (I) or a pharmaceutically acceptable saltthereof,

wherein

X is O, NH, or N-alkyl, preferably O;

Y is NH, N-alkyl, O, or C(R⁹)₂, preferably N-alkyl, O, or C(R⁹)₂;

Z is O or C(R⁹)₂, preferably C(R⁹)₂;

R¹, R², R³, and R⁴ are hydrogen;

each of R⁵, R⁶, R⁷, R⁸, and R⁹ is independently selected from hydrogen,C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl,each of which is optionally substituted with a group selected fromalkyl, amide, amine, carboxylic acid or a pharmaceutically acceptablesalt thereof, carboxyl ester, thiol, and thioether, preferably R⁵, R⁶,R⁷, and R⁸ are independently selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl,and C₁₋₆aralkyl and each R⁹ is hydrogen, more preferably, R⁶ and R⁸ areindependently C₁₋₆alkyl, R⁵ and R⁷ are independently C₁₋₆aralkyl andeach R⁹ is H;

m is an integer from 0 to 2; and

n is an integer from 0 to 2, preferably 0 or 1.

In certain embodiments, X is O and R¹, R², R³, and R⁴ are all the same,preferably R′, R², R³, and R⁴ are all hydrogen. In certain suchembodiments, R⁵, R⁶, R⁷, and R⁸ are independently selected fromC₁₋₆alkyl, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl, more preferably, R⁶ and R⁸are independently C₁₋₆alkyl and R⁵ and R⁷ are independently C₁₋₆aralkyl.

In certain preferred embodiments, X is O, R¹, R², R³, and R⁴ are allhydrogen, R⁶ and R⁸ are both isobutyl, R⁵ is phenylethyl, and R⁷ isphenylmethyl.

In certain embodiments, R⁵, R⁶, R⁷, and R⁸ are independently selectedfrom hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, andC₁₋₆aralkyl, each of which is optionally substituted with a groupselected from alkyl, amide, amine, carboxylic acid or a pharmaceuticallyacceptable salt thereof, carboxyl ester, thiol, and thioether. Incertain embodiments, at least one of R⁵ and R⁷ is C₁₋₆aralkylsubstituted with alkyl, more preferably substituted with perhaloalkyl.In certain such embodiments, R⁷ is C₁₋₆aralkyl substituted withtrifluoromethyl.

In certain embodiments, Y is selected from N-alkyl, O, and CH₂. Incertain such embodiments, Z is CH₂, and m and n are both 0. In certainalternative such embodiments, Z is CH₂, m is 0, and n is 2 or 3. In yetanother alternative such embodiments, Z is O, m is 1, and n is 2.

In certain embodiments, the invention relates to a crystalline compoundof Formula (II)

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (I) or (II), comprisingone or more of: (i) preparing the amorphous compound, e.g., according toU.S. Pat. No. 7,232,818; (ii) dissolving the amorphous compound in anorganic solvent; (iii) bringing the solution to supersaturation to causeformation of crystals; and (iv) isolating the crystals, e.g., byfiltering the crystals, by decanting fluid from the crystals, or by anyother suitable separation technique. In certain embodiments, preparationfurther comprises inducing crystallization. In certain embodiments,preparation further comprises washing the filtered crystals, e.g., witha solvent or non-solvent fluid. In certain embodiments, preparationfurther comprises drying, preferably under reduced pressure, such asunder vacuum pressure.

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (I) or (II), comprisingone or more of: (i) preparing a solution of the amorphous compound,which compound may be prepared according to, for example, U.S. Pat. No.7,232,818, in an organic solvent; (ii) bringing the solution tosupersaturation to cause formation of crystals; and (iii) isolating thecrystals, e.g., by filtering the crystals, by decanting fluid from thecrystals, or by any other suitable separation technique. In certainembodiments, preparation further comprises inducing crystallization. Incertain embodiments, preparation further comprises washing the filteredcrystals, e.g., with a solvent or non-solvent fluid. In certainembodiments, preparation further comprises drying, preferably underreduced pressure, such as under vacuum pressure.

In certain embodiments, the amorphous compound may be dissolved in anorganic solvent selected from acetonitrile, methanol, ethanol, ethylacetate, isopropanol, isopropyl acetate, isobutyl acetate, butylacetate, propyl acetate, methylethyl ketone, methylisobutyl ketone, andacetone, or any combination thereof. In certain embodiments, theamorphous compound may be dissolved in an organic solvent selected fromacetonitrile, methanol, ethanol, ethyl acetate, isopropyl acetate,methylethyl ketone, and acetone, or any combination thereof. In certainembodiments, the amorphous compound may be dissolved in an organicsolvent selected from acetonitrile, methanol, ethanol, ethyl acetate,methylethylketone, or any combination thereof. In certain embodiments,the organic solvent or solvents may be combined with water.

In certain embodiments, bringing the solution to supersaturationcomprises the addition of an anti-solvent, such as water or anotherpolar liquid miscible with the organic solvent, allowing the solution tocool, reducing the volume of the solution, or any combination thereof.In certain embodiments, bringing the solution to supersaturationcomprises adding an anti-solvent, cooling the solution to ambienttemperature or lower, and reducing the volume of the solution, e.g., byevaporating solvent from the solution. In certain embodiments, allowingthe solution to cool may be passive (e.g., allowing the solution tostand at ambient temperature) or active (e.g., cooling the solution inan ice bath or freezer).

In certain embodiments, the method further comprises inducingprecipitation or crystallization. In certain embodiments inducingprecipitation or crystallization comprises secondary nucleation, whereinnucleation occurs in the presence of seed crystals or interactions withthe environment (crystallizer walls, stirring impellers, sonication,etc.).

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, acetonitrile, methanol, ethanol,ethyl acetate, methylethyl ketone, acetone, or a combination thereof.Preferably the crystals are washed with a combination of anti-solventand the organic solvent. In certain embodiments, the anti-solvent iswater.

In certain embodiments, washing the crystals comprises washing thecrystalline compound of Formula (II) with methanol and water.

In certain embodiments, a crystalline compound of Formula (II) issubstantially pure. In certain embodiments, the melting point of thecrystalline compound of Formula (II) is in the range of about 200 toabout 220° C., about 205 to about 215° C., about 211 to about 213° C.,or even about 212° C.

In certain embodiments, the DSC of a crystalline compound of Formula(II) has a sharp endothermic maximum at about 212° C., e.g., resultingfrom melting and decomposition of the crystalline form as shown in FIG.1.

In certain embodiments, the X-ray powder pattern of a crystallinecompound of Formula (II) is (θ-2θ°): 6.10; 8.10; 9.32; 10.10; 11.00;12.14; 122.50; 13.64; 13.94; 17.14; 17.52; 18.44; 20.38; 21.00; 22.26;23.30; 24.66; 25.98; 26.02; 27.84; 28.00; 28.16; 29.98; 30.46; 32.98;33.22; 34.52; 39.46 as shown in FIG. 2.

In certain embodiments, the TG thermogram of a crystalline compound ofFormula (II) exhibits from 0.0 to 0.1% weight loss in the temperaturerange of 25 to 200° C. as shown in FIG. 3.

In certain embodiments, a crystalline compound of Formula (II) is notsolvated (e.g., the crystal lattice does not comprise molecules of asolvent). In certain alternative embodiments, a crystalline compound ofFormula (II) is solvated.

In certain embodiments, the invention relates to a method for thepreparation of an amorphous compound of Formula (II) comprising one ormore of (i) dissolving the crystalline compound in an organic solvent;(ii) bringing the solution to supersaturation to cause formation ofcrystals; and (iii) isolating the crystals, e.g., by filtering thecrystals, by decanting fluid from the crystals, or by any other suitableseparation technique. In certain embodiments, preparation furthercomprises inducing precipitation. In certain embodiments, preparationfurther comprises washing the amorphous compound. In certainembodiments, the method further comprises drying, preferably underreduced pressure, such as under vacuum pressure. In certain embodiments,the invention relates to a crystalline salt of a compound of Formula (I)or (II), wherein the salt counterion is selected from bromide, chloride,sulfate, phosphate, nitrate, acetate, trifluoroacetate, citrate,methanesulfonate, valerate, oleate, palmitate, stearate, laurate,benzoate, lactate, succinate, tosylate, malonate, maleate, fumarate,succinate, tartrate, mesylate, 2-hydroxyethansulfonate, and the like. Incertain such embodiments, the salt counterion is selected from citrate,tartrate, trifluoroacetate, methanesulfonate, toluenesulfonate,chloride, and bromide, preferably citrate.

In certain embodiments, the invention relates to a method for thepreparation of a crystalline salt of a compound of Formula (II),comprising one or more of: (i) preparing the amorphous compound e.g.,according to U.S. Pat. No. 7,232,818; (ii) dissolving the amorphouscompound in an organic solvent; (iii) bringing the solution tosupersaturation to cause formation of crystals; and (iv) isolating thecrystals, e.g., by filtering the crystals, by decanting fluid from thecrystals, or by any other suitable separation technique. In certainembodiments, preparation further comprises inducing crystallization. Incertain embodiments, preparation further comprises washing the crystals,e.g., with a solvent or non-solvent fluid. In certain embodiments,preparation further comprises drying, preferably under reduced pressure,such as under vacuum pressure.

In certain embodiments, the invention relates to a method for thepreparation of a crystalline salt of a compound of Formula (II),comprising one or more of (i) preparing a solution of a compound ofFormula (II) in an organic solvent; (ii) adding a suitable acid; (iii)bringing the solution to supersaturation to cause formation of crystals;and (iv) isolating the crystals, e.g., by filtering the crystals, bydecanting fluid from the crystals, or by any other suitable separationtechnique. In certain embodiments, preparation further comprisesinducing crystallization. In certain embodiments, preparation furthercomprises washing the crystals, e.g., with a solvent or non-solventfluid. In certain embodiments, preparation further comprises drying,preferably under reduced pressure, such as under vacuum pressure. Incertain embodiments where the salt is less soluble in a solvent than thefree base, adding the acid to a solution may itself be sufficient tobring the solution to supersaturation.

In certain embodiments, the salt counterion is selected from selectedfrom bromide, chloride, sulfate, phosphate, nitrate, acetate,trifluoroacetate, citrate, methanesulfonate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, succinate, tosylate,malonate, maleate, fumarate, succinate, tartrate, mesylate,2-hydroxyethansulfonate, and the like. In certain such embodiments, thesalt counterion is selected from citrate, tartrate, trifluoroacetate,methanesulfonate, toluenesulfonate, chloride, and bromide, preferablycitrate.

In certain embodiments, the organic solvent is selected from THF,acetonitrile, ether, and MTBE, or any combination thereof, preferablyTHF or acetonitrile, or a combination thereof.

In certain embodiments, a crystalline citrate salt of a compound ofFormula (II) is substantially pure. In certain embodiments, the meltingpoint of the crystalline citrate salt of a compound of Formula (II) isin the range of about 180 to about 190° C. or even about 184 to about188° C.

In certain embodiments, the DSC of a crystalline citrate salt of acompound of Formula (II) has a sharp endothermic maximum at about 187°C., e.g., resulting from melting and decomposition of the crystallineform as shown in FIG. 11.

In certain embodiments, the X-ray powder pattern of a crystallinecitrate salt of a compound of Formula (II) is (θ-2θ°): 4.40; 7.22; 9.12;12.36; 13.35; 14.34; 15.54; 16.14; 16.54; 17.00; 18.24; 18.58; 19.70;19.90; 20.30; 20.42; 21.84; 22.02; 23.34; 23.84; 24.04; 24.08; 24.48;24.76; 25.48; 26.18; 28.14; 28.20; 28.64; 29.64; 31.04; 31.84; 33.00;33.20; 34.06; 34.30; 34.50; 35.18; 37.48; 37.90; 39.48 as shown in FIG.12.

In certain embodiments, the invention relates to a crystalline compoundof Formula (III)

wherein X is any suitable counterion.

In certain embodiments, X is a counterion selected from bromide,chloride, sulfate, phosphate, nitrate, acetate, trifluoroacetate,citrate, methanesulfonate, valerate, oleate, palmitate, stearate,laurate, benzoate, lactate, succinate, tosylate, malonate, maleate,fumarate, succinate, tartrate, mesylate, 2-hydroxyethansulfonate, andthe like. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”,J. Pharm. Sci. 66: 1-19.) In certain embodiments X is selected fromtrifluoroacetate, methanesulfonate, toluenesulfonate, acetate, chloride,and bromide, preferably trifluoroacetate.

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (III) comprising one ormore of: (i) preparing a compound of Formula (IV), e.g., according toBioorg. Med. Chem. Letter 1999, 9, 2283-88 or U.S. Patent Application2005-0256324, wherein PG is a suitable protecting group (e.g., Boc orCbz)

(ii) dissolving the compound of Formula (IV) in an organic solvent;(iii) adding a suitable acid; (iv) bringing the solution tosupersaturation to cause formation of crystals; and (v) isolating thecrystals, e.g., by filtering the crystals, by decanting fluid from thecrystals, or by any other suitable separation technique. In certainembodiments, preparation further comprises inducing crystallization. Incertain embodiments, preparation further comprises washing the crystals,e.g., with a solvent or non-solvent fluid. In certain embodiments,preparation further comprises drying, preferably under reduced pressure,such as under vacuum pressure.

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (III), comprising oneor more of: (i) preparing a solution of an amorphous compound of Formula(IV), e.g., according to Bioorg. Med. Chem. Letter 1999, 9, 2283-88 orU.S. Patent Application 2005-0256324, in an organic solvent, wherein PGis a suitable protecting group (e.g., Boc or Cbz).

(ii) bringing the solution to supersaturation to cause formation ofcrystals; and (iii) isolating the crystals, e.g., by filtering thecrystals, by decanting fluid from the crystals, or by any other suitableseparation technique. In certain embodiments, preparation furthercomprises inducing crystallization. In certain embodiments, preparationfurther comprises washing the crystals, e.g., with a solvent ornon-solvent fluid. In certain embodiments, preparation further comprisesdrying, preferably under reduced pressure, such as under vacuumpressure.

In certain embodiments the acid is selected from hydrobromic,hydrochloric, sulfuric, phosphoric, nitric, acetic, trifluoroacetic,citric, methanesulfonic, valeric, oleaic, palmitic, stearic, lauric,benzoic, lactic, succinic, p-toluenesulfonic, citric, malonic, maleic,fumaric, succinic, tartaric, methanesulfonic, 2-hydroxyethanesulfonic,and the like. Preferably the acid is trifluoroacetic acid.

In certain embodiments, X is a counterion selected from hydrobromide,hydrochloride, sulfate, phosphate, nitrate, acetate, trifluoroacetate,citrate, methanesulfonate, valerate, oleate, palmitate, stearate,laurate, benzoate, lactate, succinate, tosylate, malonate, maleate,fumarate, succinate, tartrate, mesylate, 2-hydroxyethansulfonate, andthe like. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”,J. Pharm. Sci. 66: 1-19.) In certain embodiments, X is selected fromtrifluoroacetate, methanesulfonate, toluenesulfonate, acetate, chloride,and bromide, preferably trifluoroacetate.

In certain embodiments, the compound of Formula (IV) may be dissolved inan organic solvent selected from dichloromethane, ethyl acetate,isopropyl acetate, isobutyl acetate, butyl acetate, propyl acetate,diethyl ether, methyl tert-butyl ether (MTBE), or any combinationthereof. In certain embodiments, the organic solvent is selected fromdichloromethane, ethyl acetate, MTBE, or any combination thereof,preferably either dichloromethane and MTBE or ethyl acetate and MTBE.

In certain embodiments, bringing the solution to supersaturationcomprises the addition of an anti-solvent, such as hexanes or heptanesor another liquid miscible with the organic solvent, allowing thesolution to cool, reducing the volume of the solution, or anycombination thereof. In certain embodiments, bringing the solution tosupersaturation comprises adding an anti-solvent, cooling the solutionto ambient temperature or lower, and reducing the volume of thesolution, e.g., by evaporating solvent from the solution. In certainembodiments, the anti-solvent is hexanes or heptanes, preferablyheptanes.

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, ethyl acetate, dichloromethane, or acombination thereof. Preferably the crystals are washed withanti-solvent, preferably heptanes.

In certain embodiments, the DSC of a crystalline compound of Formula(III) has a sharp endothermic maximum at about 137° C., e.g., resultingfrom melting and decomposition of the crystalline form as shown in FIG.9.

In certain embodiments, the X-ray powder pattern of a crystallinecompound of Formula (II) is (θ-2θ°): 8.84; 15.18; 15.32; 16.20; 16.82;17.66; 18.26; 19.10; 21.20; 22.58; 23.06; 23.52; 25.32; 26.58; 28.60;30.08; 30.48; 30.84; 32.20; 36.14; 37.12 as shown in FIG. 10.

In certain embodiments, a crystalline compound of Formula (III) is notsolvated (e.g., the crystal lattice does not comprise molecules of asolvent). In certain alternative embodiments, a crystalline compound ofFormula (III) is solvated.

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (II), comprising one ormore of (i) preparing a solution of compound of Formula (IV) wherein PGis a suitable protecting group (e.g., Boc or Cbz), in a first organicsolvent

(ii) adding a suitable acid; (iii) bringing the solution tosupersaturation to cause formation of crystals; (iv) isolating thecrystals to provide a crystalline compound of Formula (III); (v)reacting the crystalline compound of Formula (III)

wherein X is any suitable counterion, with a compound of Formula (V)

to provide a compound of Formula (II); (vi) preparing a solution of thecompound of Formula (II) in a second organic solvent; (vii) bringing thesolution to supersaturation to cause formation of crystals; and (viii)isolating the crystals to provide a crystalline compound of Formula(II), e.g., by filtering the crystals, by decanting, or by any othersuitable separation technique. In certain embodiments, preparationfurther comprises inducing crystallization. In certain embodiments,preparation further comprises washing the crystals, e.g., with a solventor non-solvent fluid. In certain embodiments, preparation furthercomprises drying, preferably under reduced pressure, such as undervacuum pressure.

In certain embodiments, the acid is selected from hydrobromic,hydrochloric, sulfuric, phosphoric, nitric, acetic, trifluoroacetic,citric, methanesulfonic, valeric, oleaic, palmitic, stearic, lauric,benzoic, lactic, succinic, p-toluenesulfonic, citric, malonic, maleic,fumaric, succinic, tartaric, methanesulfonic, 2-hydroxyethansulfonic,and the like. Preferably the acid is trifluoroacetic acid.

In certain embodiments, X is a counterion selected from hydrobromide,hydrochloride, sulfate, phosphate, nitrate, acetate, trifluoroacetate,citrate, methanesulfonate, valerate, oleate, palmitate, stearate,laurate, benzoate, lactate, succinate, tosylate, malonate, maleate,fumarate, succinate, tartrate, mesylate, 2-hydroxyethanesulfonate, andthe like. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”,J. Pharm. Sci. 66: 1-19.) In certain embodiments, X is selected fromtrifluoroacetate, methanesulfonate, toluenesulfonate, acetate, chloride,and bromide, preferably trifluoroacetate.

In certain embodiments, the first organic solvent is selected fromdichloromethane, ethyl acetate, isopropyl acetate, isobutyl acetate,butyl acetate, propyl acetate, diethyl ether, methyl tert-butyl ether(MTBE), or any combination thereof. In certain embodiments, the organicsolvent is selected from dichloromethane, ethyl acetate, MTBE, or anycombination thereof, preferably either dichloromethane and MTBE or ethylacetate and MTBE.

In certain embodiments, the second organic solvent is selected fromacetonitrile, methanol, ethanol, ethyl acetate, isopropanol, isopropylacetate, isobutyl acetate, butyl acetate, propyl acetate, methylethylketone, methylisobutyl ketone, and acetone, or any combination thereof.In certain embodiments, the amorphous compound may be dissolved in anorganic solvent selected from acetonitrile, methanol, ethanol, ethylacetate, acetone, or any combination thereof. In certain embodiments,the organic solvent or solvents may be combined with water.

In certain embodiments, preparation further comprises washing thecrystals of either or both of Formula (II) or (III). In certainembodiments, washing the crystals of a compound of Formula (II)comprises washing with a liquid selected from anti-solvent,acetonitrile, methanol, ethanol, ethyl acetate, acetone, or acombination thereof. Preferably the crystals of a compound of Formula(II) are washed with a combination of anti-solvent and the organicsolvent. In certain embodiments, washing the crystals comprises washingthe crystalline compound of Formula (II) with methanol and water. Incertain embodiments, washing the crystals of a compound of Formula (III)comprises washing with a liquid selected from anti-solvent, ethylacetate, dichloromethane, or a combination thereof. Preferably thecrystals of a compound of Formula (III) are washed with anti-solvent,preferably heptanes.

In certain embodiments, preparation further comprises drying thecrystals of either or both of Formula (II) or (III), preferably underreduced pressure, such as under vacuum pressure.

In certain embodiments, the invention relates to a pharmaceuticalcomposition comprising a crystalline compound of Formula (I) or (II) anda pharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical composition is selected from tablets, capsules, andinjections.

This invention also relates to methods for the synthesis ofepoxyketones, such as formulae (III) and (IV) above. Thus, in anotheraspect, the invention provides a method for preparing amino acidketo-epoxides according to scheme (I)

wherein

-   R¹ is selected from a protecting group or a further chain of amino    acids, which itself may be optionally substituted, preferably a    protecting group, most preferably an electron withdrawing protecting    group;-   R² is selected from hydrogen and C₁₋₆alkyl; and-   R³ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkoxyalkyl,    heterocyclyl, aryl, heteroaryl, C₁₋₆heteroaralkyl, and C₁₋₆aralkyl;    and-   wherein the method comprises a stereoselective epoxidation under    epoxidizing conditions, preferably an aqueous sodium hypochlorite    (bleach) or calcium hypochlorite solution in the presence of a    cosolvent selected from pyridine, acetonitrile, DMF, DMSO, NMP, DMA,    THF, and nitromethane.

In certain embodiments, the cosolvent is selected from NMP and pyridine,preferably pyridine.

In certain embodiments, the epoxidation is performed using aqueoussodium hypochlorite in the presence of a cosolvent selected frompyridine, acetonitrile, DMF, DMSO, NMP, DMA, THF, and nitromethane,preferably NMP or pyridine, more preferably pyridine. In certainembodiments, the epoxidation is performed using a 10% aqueous sodiumhypochlorite solution. In certain embodiments, the epoxidation isperformed using a 10% aqueous sodium hypochlorite solution in thepresence of pyridine. In certain embodiments, the epoxidation isperformed using a calcium hypochlorite solution in the presence of NMP.

In certain embodiments, R¹ is selected from a protecting group or afurther chain of amino acids, which itself may be optionallysubstituted. In certain such embodiments, R¹ is a protecting group,preferably an electron withdrawing protecting group.

In certain embodiments, R¹ is selected from t-butoxy carbonyl (Boc),benzoyl (Bz), fluoren-9-ylmethoxycarbonyl (Fmoc),trichloroethoxycarbonyl (Troc), and benzyloxy carbonyl (Cbz). In certainsuch embodiments, R¹ is selected from t-butoxy carbonyl (Boc), benzoyl(Bz), trichloroethoxycarbonyl (Troc), and benzyloxy carbonyl (Cbz),preferably Cbz or Boc. In certain preferred embodiments, R¹ is Boc.

In certain embodiments, R³ is selected from hydrogen, C₁₋₆alkyl,C₁₋₆alkoxyalkyl, heterocyclyl, aryl, heteroaryl, C₁₋₆heteroaralkyl, andC₁₋₆aralkyl. In preferred embodiments, R³ is C₁₋₆alkyl, preferablyisobutyl. In certain preferred embodiments, R³ is C₁₋₆aralkyl,preferably phenylmethyl, 4-hydroxyphenylmethyl, or 2-phenylethyl.

In certain embodiments, the stereoselective epoxidation is performedunder conditions that do not result in significant epimerization of thecarbon bearing R³, such that there is less than 10%, less than 5%, lessthan 2%, or even less than 1% epimerization of the carbon bearing R³. Incertain embodiments, the stereoselective epoxidation is performed suchthat the product is greater than about 90%, greater than 95%, greaterthan 98%, or even greater than 99% diastereomerically pure.

In certain embodiments, the epoxidation is performed at a temperature inthe range of about −15° C. to about 10° C., about −10° C. to about 5°C., or even about −5° C. to about 0° C.

In certain embodiments, the compounds in scheme I have the followingstereochemistry

In certain embodiments, the stereoselective epoxidation is performedsuch that the product is greater than about 90%, greater than 95%,greater than 98%, or even greater than 99% diastereomerically pure.

The use of various N-protecting groups, e.g., the benzyloxy carbonylgroup or the t-butyloxycarbonyl group (Boc), various coupling reagents,e.g., dicyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC),N-hydroxyazabenzotriazole (HATU), carbonyldiimidazole, or1-hydroxybenzotriazole monohydrate (HOBT), and various cleavageconditions: for example, trifluoracetic acid (TFA), HCl in dioxane,hydrogenation on Pd/C in organic solvents (such as methanol or ethylacetate), boron tris(trifluoroacetate), and cyanogen bromide, andreaction in solution with isolation and purification of intermediatesare well-known in the art of peptide synthesis, and are equallyapplicable to the preparation of the subject compounds (Greene, T. W.;Wuts, P. G. M. Protective Groups in Organic Synthesis, 3^(rd) ed.;Wiley: New York, 1999).

In certain embodiments, the amino acid keto-epoxide may be furthermodified by deprotection of the amine, if applicable, and coupling witha chain of amino acids. Methods for the coupling of such fragments arewell known in the art (Elofsson, M., et al. (1999) Chemistry & Biology,6:811-822; Elofsson, M., et al (1999) Chemistry & Biology, 6:811-822).In a preferred embodiment, the chain of amino acids comprises one tothree amino acids.

In certain embodiments, the chain of amino acids has a structure offormula (VI) or a pharmaceutically acceptable salt thereof

-   wherein each A is independently selected from C═O, C═S, and SO₂,    preferably C═O; or-   A is optionally a covalent bond when adjacent to an occurrence of Z;-   L is absent or is selected from C═O, C═S, and SO₂, preferably L is    absent or C═O;-   M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;-   Q is absent or is selected from O, NH, and N—C₁₋₆alkyl, preferably Q    is absent, O, or NH, most preferably Q is absent or O;-   X is COOH or an activated form thereof, preferably X is COOH, COCl,    or CON(Me)(OMe), most preferably X is COOH or COCl;-   Y is absent or is selected from O, NH, N—C₁₋₆alkyl, S, SO, SO₂,    CHOR¹⁷, and CHCO₂R¹⁷;-   each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,    preferably O; or-   Z is optionally a covalent bond when adjacent to an occurrence of A;-   R⁵, R⁶, and R⁷ are each independently selected from C₁₋₆alkyl,    C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of    which is optionally substituted with one or more of amide, amine,    carboxylic acid (or a salt thereof), ester (including C₁₋₆alkyl and    C₁₋₅alkyl ester and aryl ester), thiol, or thioether substituents;-   R⁹ is N(R¹⁰)LQR¹¹;-   R¹⁰, R¹², and R¹³ are independently selected from hydrogen, OH, and    C₁₋₆alkyl, preferably, R¹⁰ is selected from hydrogen, OH, and    C₁₋₆alkyl, and R¹² and R¹³ are independently selected from hydrogen    and C₁₋₆alkyl, preferably hydrogen;-   R¹¹ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,    aryl, C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹⁵ZAZ—C₁₋₈alkyl-,    R¹⁸Z—C₁₋₈alkyl-, (R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    R¹⁵ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-, (R¹⁷)₂N—C₁₋₁₂alkyl-,    (R¹⁷)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-,    R¹⁸SO₂C₁₋₈alkyl-, and R¹⁸SO₂NH; preferably C₁₋₆alkyl, C₁₋₆alkenyl,    C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl,    R¹⁵ZA-C₁₋₈alkyl-, R¹⁸Z—C₁₋₈alkyl-,    (R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    (R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,    R¹⁵ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-, (R¹⁷)₂N—C₁₋₈alkyl-,    (R¹⁷)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,    R¹⁸SO₂C₁₋₈alkyl-, and R¹⁸SO₂NH, wherein each occurrence of Z and A    is independently other than a covalent bond; or-   R¹⁰ and R¹¹ together are C₁₋₆alkyl-Y—C₁₋₆alkyl,    C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,    ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, thereby forming a ring;    preferably C₁₋₂alkyl-Y—C₁₋₂alkyl, C₁₋₂alkyl-ZA-C₁₋₂alkyl,    A-C₁₋₂alkyl-ZA-C₁₋₂alkyl, A-C₁₋₃alkyl-A, or C₁₋₄alkyl-A, wherein    each occurrence of Z and A is independently other than a covalent    bond;-   R¹⁵ and R¹⁶ are independently selected from hydrogen, metal cation,    C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl,    and C₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, and    C₁₋₆alkyl, or R¹⁵ and R¹⁶ together are C₁₋₆alkyl, thereby forming a    ring;-   each R¹⁷ is independently selected from hydrogen and C₁₋₆alkyl,    preferably C₁₋₆alkyl;-   R¹⁸ is independently selected from hydrogen, OH, C₁₋₆alkyl,    C₁₋₆alkenyl, C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl,    heteroaryl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl;-   provided that in any occurrence of the sequence ZAZ, at least one    member of the sequence must be other than a covalent bond.

In some embodiments, R⁵, R⁶, and R⁷ are selected from C₁₋₆alkyl orC₁₋₆aralkyl. In preferred embodiments, R⁶ is C₁₋₆alkyl and R⁵ and R⁷ areC₁₋₆aralkyl. In the most preferred embodiment, R⁶ is isobutyl, R⁵ is2-phenylethyl, and R⁷ is phenylmethyl.

In certain embodiments, L and Q are absent and R¹¹ is selected fromC₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.In certain such embodiments, R¹⁰ is C₁₋₆alkyl and R¹¹ is selected frombutyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R¹¹ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R¹¹ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R¹¹ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R¹⁵ZA-C₁₋₈alkyl-, R¹⁸Z—C₁₋₈alkyl-,(R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-,(R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,R¹⁵ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹⁷)₂N—C₁₋₈alkyl-, (R¹⁷)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,R¹⁸SO₂C₁₋₈alkyl-, and R¹⁸SO₂NH—, wherein each occurrence of Z and A isindependently other than a covalent bond. In certain embodiments, L isC═O, Q is absent, and R¹¹ is H.

In certain embodiments, R¹⁰ is C₁₋₆alkyl, R¹¹ is C₁₋₆alkyl, Q is absent,and L is C═O In certain such embodiments, R¹¹ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R¹¹ is C₁₋₆aralkyl. Incertain such embodiments, R¹¹ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R¹⁰ is C₁₋₆alkyl, and R¹¹is aryl. In certain such embodiments, R¹¹ is substituted orunsubstituted phenyl.

In certain embodiments, L is C═O, Q is absent or O, n is 0 or 1, and R¹¹is —(CH₂)_(n)carbocyclyl. In certain such embodiments, R¹¹ iscyclopropyl or cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, n is aninteger from 1 to 8 (preferably 1), and R¹¹ is selected fromR¹⁵ZA-C₁₋₈alkyl-, R¹⁸Z—C₁₋₈alkyl-, R¹⁵ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹⁵O)(R¹⁶O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-, wherein each occurrence of A isindependently other than a covalent bond. In certain such embodiments,R⁷ is heterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R¹²)(R¹³), wherein R¹² and R¹³ togetherare C₁₋₆alkyl-Y—C₁₋₆ alkyl, preferably C₁₋₃ alkyl-Y—C₁₋₃ alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, n is an integerfrom 1 to 8, and R¹¹ is selected from (R¹⁵O)(R¹⁶O)F(O)O—C₁₋₈alkyl-,(R¹⁷)₂NC₁₋₈alkyl, (R¹⁷)₃N⁺(CH₂)_(n)—, and heterocyclyl-M-. In certainsuch embodiments, R¹¹ is —C₁₋₈alkylN(R¹⁷)₂ or —C₁₋₈alkylN⁺(R¹⁷)₃, whereR¹⁷ is C₁₋₆alkyl. In certain other such embodiments, R¹¹ isheterocyclylM-, where heterocyclyl is selected from morpholino,piperidino, piperazino, and pyrrolidino.

In certain embodiments, L is C═O, R¹⁰ is C₁₋₆alkyl, Q is selected from Oand NH and R¹¹ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl. In other embodiments, L is C═O, R¹⁰ is C₁₋₆alkyl,Q is selected from O and NH, and R¹¹ is C₁₋₆alkyl, where C₁₋₆alkyl isselected from methyl, ethyl, and isopropyl. In further embodiments, L isC═O, R¹⁰ is C₁₋₆alkyl, Q is selected from O and NH and R¹¹ isC₁₋₆aralkyl, where aralkyl is phenylmethyl. In other embodiments, L isC═O, R¹⁰ is C₁₋₆alkyl, Q is selected from O and NH, and R¹¹ isC₁₋₆heteroaralkyl, where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R¹⁰ and R¹¹ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,wherein each occurrence of Z and A is independently other than acovalent bond, thereby forming a ring. In certain preferred embodiments,L is C═O, Q and Y are absent, and R¹⁰ and R¹¹ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and Q areabsent, and R¹⁰ and R¹¹ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In anotherpreferred embodiment, L is C═O, Q is absent, Y is selected from NH andN—C₁₋₆alkyl, and R¹⁰ and R¹¹ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L is C═O, Y is absent, and R¹⁰ and R¹¹together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Land A are C═O, and R¹⁰ and R¹¹ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. Inanother preferred embodiment, L and A are C═O and R¹⁰ and R¹¹ togetherare C₂₋₃alkyl-A.

In certain embodiments, the chain of amino acids has a structure offormula (VII)

-   wherein-   each A is independently selected from C═O, C═S, and SO₂, preferably    C═O; or-   A is optionally a covalent bond when adjacent to an occurrence of Z;-   each B is independently selected from C═O, C═S, and SO₂, preferably    C═O;-   D is absent or is C₁₋₈alkyl;-   G is selected from O, NH, and N—C₁₋₆alkyl;-   K is absent or is selected from C═O, C═S, and SO₂, preferably K is    absent or is C═O;-   L is absent or is selected from C═O, C═S, and SO₂, preferably L is    absent or C═O;-   M is absent or is C₁₋₈alkyl;-   Q is absent or is selected from O, NH, and N—C₁₋₆alkyl, preferably Q    is absent, O, or NH, most preferably Q is absent;-   X is COOH or an activated form thereof, preferably X is COOH, COCl,    or CON(Me)(OMe), most preferably X is COOH or COCl;-   each V is independently absent or is selected from O, S, NH, and    N—C₁₋₆alkyl, preferably V is absent or O;-   W is absent or is independently selected from O, S, NH, and    N—C₁₋₆alkyl, preferably O;-   Y is absent or is selected from O, NH, N—C₁₋₆alkyl, S, SO, SO₂,    CHOR¹⁷, and CHCO₂R¹⁷;-   each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,    preferably O; or-   Z is optionally a covalent bond when adjacent to an occurrence of A;-   R⁵, R⁶, and R⁷ are each independently selected from C₁₋₆alkyl,    C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, C₁₋₆aralkyl, and    R¹⁶DVKOC₁₋₃alkyl-, wherein at least one of R⁵ and R⁷ is    R¹⁶DVKOC₁₋₃alkyl-;-   R⁹ is N(R¹⁰)LQR¹¹;-   R¹⁰ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably    hydrogen or C₁₋₆alkyl;-   R¹¹ is a further chain of amino acids, hydrogen, a protecting group,    aryl, or heteroaryl, any of which is optionally substituted with    halogen, carbonyl, nitro, hydroxy, aryl, C₁₋₅alkyl; or R¹¹ is    selected from C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl,    C₁₋₆heteroaralkyl, R¹²ZAZ—C₁₋₈alkyl-, R¹⁵ZAZ—C₁₋₈alkyl-,    (R¹²O)(R¹³O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    R¹²ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R¹²O)(R¹³O)P(═O)O—C₁₋₈alkyl-, (R¹⁴)₂N—C₁₋₈alkyl-,    (R¹⁴)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,    R¹⁵SO₂C₁₋₈alkyl-, and R¹⁵SO₂NH; or-   R¹⁰ and R¹¹ together are C₁₋₆alkyl-Y—C₁₋₆alkyl,    C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,    ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-ZAZ;-   R¹² and R¹³ are independently selected from hydrogen, metal cation,    C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl,    and C₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, and    C₁₋₆alkyl, or R¹² and R¹³ together are C₁₋₆alkyl, thereby forming a    ring;-   each R¹⁴ is independently selected from hydrogen and C₁₋₆alkyl,    preferably C₁₋₆alkyl;-   each R¹⁵ is independently selected from hydrogen, OR¹⁴, C₁₋₆alkyl,    C₁₋₆alkenyl, C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl,    heteroaryl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl;-   R¹⁶ is selected from hydrogen, (R¹⁷O)(R¹⁸O)P(═O)W—, R¹⁷GB-,    heterocyclyl-, (R¹⁹)₂N—, (R¹⁹)₃N⁺—, R¹⁹SO₂ GBG-, and R¹⁷    GBC₁₋₈alkyl- where the C₁₋₈alkyl moiety is optionally substituted    with OH, C₁₋₈alkylW (optionally substituted with halogen, preferably    fluorine), aryl, heteroaryl, carbocyclyl, heterocyclyl, and    C₁₋₆aralkyl, preferably at least one occurrence of R¹⁶ is other than    hydrogen;-   R¹⁷ and R¹⁸ are independently selected from hydrogen, metal cation,    C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl,    and C₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, and    C₁₋₆alkyl, or R¹⁷ and R¹⁸ together are C₁₋₆alkyl, thereby forming a    ring; and-   each R¹⁹ is independently selected from hydrogen, OR¹⁴, C₁₋₆alkyl,    C₁₋₆alkenyl, C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl,    heteroaryl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl; and-   D, G, V, K, and W are selected such that there are no O—O, N—O, S—N,    or S—O bonds.

In certain embodiments, R⁵, R⁶, and R⁷ are each independently selectedfrom C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, C₁₋₆aralkyl,and R¹⁶DVKOC₁₋₃alkyl- wherein at least one of R⁵ and R⁷ isR¹⁶DVKOC₁₋₃alkyl-. In preferred embodiments, one of R⁵ and R⁷ isC₁₋₆aralkyl and the other is R¹⁶DVKOC₁₋₃alkyl-, and R⁶ is independentlyC₁₋₆alkyl. In the most preferred embodiment, one of R⁵ and R⁷ is2-phenylethyl or phenylmethyl and the other is R¹⁶DVKOCH₂— orR¹⁶DVKO(CH₃)CH—, and R⁶ is isobutyl.

In certain embodiments, each R¹⁵ is independently selected fromhydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.

In certain embodiments, each R¹⁹ is independently selected fromhydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.

In certain embodiments, L and Q are absent and R¹¹ is selected fromhydrogen, a further chain of amino acids, C₁₋₆acyl, a protecting group,aryl, heteroaryl, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl. In certain such embodiments, R¹⁰ is C₁₋₆alkyl and R¹¹is selected from butyl, allyl, propargyl, phenylmethyl, 2-pyridyl,3-pyridyl, and 4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R¹¹ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R¹¹ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R¹¹ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R¹²ZA-C₁₋₈alkyl-, R¹⁵Z—C₁₋₈alkyl-,(R¹²O)(R¹³O)P(═O)O—C₁₋₈alkyl-,(R¹²O)(R¹³O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹²O)(R¹³O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, R¹²ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-, (R¹⁴)₂N—C₁₋₈alkyl-,(R¹⁴)₃N⁺-C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-, R¹⁵SO₂C₁₋₈alkyl-,and R¹⁵SO₂NH—. In certain embodiments, L is C═O, Q is absent, and R¹¹ isH.

In certain embodiments, R¹⁰ is C₁₋₆alkyl, R¹¹ is C₁₋₆alkyl, Q is absent,and L is C═O. In certain such embodiments, R¹¹ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R¹¹ is C₁₋₆aralkyl. Incertain such embodiments, R¹¹ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R¹⁰ is C₁₋₆alkyl, and R¹¹is aryl. In certain such embodiments, R¹¹ is substituted orunsubstituted phenyl.

In certain embodiments, L is C═O, Q is absent or O, and R¹¹ is—(CH₂)_(n)carbocyclyl. In certain such embodiments, R¹¹ is cyclopropylor cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, and R¹¹ isselected from R¹²ZA-C₁₋₈alkyl-, R¹⁵Z—C₁₋₈alkyl-,R¹²ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹²O)(R¹³O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹²O)(R¹³O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-. In certain such embodiments, R¹¹ isheterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R²⁰)(R²¹), wherein R²⁰ and R²¹ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, preferably C₁₋₃alkyl-Y—C₁₋₃alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, and R¹¹ isselected from (R¹²O)(R¹³O)P(═O)O—C₁₋₈alkyl-, (R¹⁴)₂NC₁₋₈alkyl,(R¹⁴)₃N⁺(CH₂)_(n)—, and heterocyclyl-M-. In certain such embodiments,R¹¹ is —C₁₋₈alkylN(R¹⁴)₂ or —C₁₋₈alkylN⁺(R¹⁴)₃, where R¹⁴ is C₁₋₆alkyl.In certain other such embodiments, R¹¹ is heterocyclylM-, whereheterocyclyl is selected from morpholino, piperidino, piperazino, andpyrrolidino.

In certain embodiments, L is C═O, R¹⁰ is C₁₋₆alkyl, Q is selected from Oand NH and R¹¹ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆araalkyl,and C₁₋₆heteroaraalkyl. In other embodiments, L is C═O, R¹⁰ isC₁₋₆alkyl, Q is selected from O and NH, and R¹¹ is C₁₋₆alkyl, whereC₁₋₆alkyl is selected from methyl, ethyl, and isopropyl. In furtherembodiments, L is C═O, R¹⁰ is C₁₋₆alkyl, Q is selected from O and NH andR¹¹ is C₁₋₆aralkyl, where aralkyl is phenylmethyl. In other embodiments,L is C═O, R¹⁰ is C₁₋₆alkyl, Q is selected from O and NH, and R¹¹ isC₁₋₆heteroaralkyl, where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R¹⁰ and R¹¹ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,thereby forming a ring. In certain preferred embodiments, L is C═O, Qand Y are absent, and R¹⁰ and R¹¹ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L and Q are absent, and R¹⁰ and R¹¹together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Lis C═O, Q is absent, Y is selected from NH and N—C₁₋₆alkyl, and R¹⁰ andR¹¹ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment,L is C═O, Y is absent, and R¹⁰ and R¹¹ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and A are C═O,and R¹⁰ and R¹¹ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. In anotherpreferred embodiment, L and A are C═O and R¹⁰ and R¹¹ together areC₂₋₃alkyl-A.

In certain embodiments, R¹⁶ is (R¹⁷O)(R¹⁸O)P(═O)W—. In certain suchembodiments, D, V, K, and W are absent. In other such embodiments, V andK are absent, D is C₁₋₈alkyl, and W is O. In yet other such embodiments,D is C₁₋₈alkyl, K is C═O, and V and W are O.

In certain embodiments, R¹⁶ is R¹⁷GB-. In preferred embodiments, B isC═O, G is O, D is C₁₋₈alkyl, V is 0, and K is C═O.

In certain embodiments, R¹⁶ is heterocyclyl-. In preferred suchembodiments, D is C₁₋₈alkyl. In certain such embodiments, V is O, K isC═O, and heterocyclyl is oxodioxolenyl. In other such embodiments, V isabsent, K is absent or is C═O, and heterocyclyl is N(R²⁰)(R²¹), whereR²⁰ and R²¹ together are J-T-J, J-WB-J, or B-J-T-J, T is absent or isselected from O, NR¹⁷, S, SO, SO₂, CHOR¹⁹, CHCO₂R¹⁷, C═O, CF₂, and CHF,and J is absent or is C₁₋₃alkyl.

In certain embodiments, R¹⁶ is (R¹⁹)₂N— or (R¹⁹)₃N⁺—, and preferably Vis absent. In preferred such embodiments, D is C₁₋₈alkyl and K is absentor C═O. In certain embodiments where V is absent and R¹⁶ is (R¹⁹)₂N—, Dis absent K is absent or is C═O, preferably K is C═O.

In certain embodiments, R¹⁶ is R¹⁹SO₂ GBG-. In preferred suchembodiments, B is C═O, D, V, and K are absent, and G is NH orNC₁₋₆alkyl.

In certain embodiments, R¹⁶ is R¹⁷ GBC₁₋₈alkyl-. In preferredembodiments, B is C═O, G is O, and the C₁₋₈alkyl moiety is optionallysubstituted with OH, C₁₋₈alkyl (optionally substituted with halogen,preferably fluorine), C₁₋₈alkylW, aryl, heteroaryl, carbocyclyl,heterocyclyl, and C₁₋₆aralkyl. In certain such embodiments, theC₁₋₈alkyl moiety is an unsubstituted, mono-, or disubstituted C₁alkyl.

In certain embodiments, the chain of amino acids has a structure offormula (VIII) or (IX) or a pharmaceutically acceptable salt thereof

wherein

-   each Ar is independently an aromatic or heteroaromatic group    optionally substituted with 1 to 4 substituents;-   L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ or    C═O;-   X is COOH or an activated form thereof, preferably X is COOH, COCl,    or CON(Me)(OMe), most preferably X is COOH or COCl;-   Y is absent or is selected from C═O and SO₂;-   Z is absent or is C₁₋₆alkyl;-   R⁵ and R⁶ are each independently selected from C₁₋₆alkyl,    C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of    which is optionally substituted with one or more of amide, amine,    carboxylic acid (or a salt thereof), ester (including C₁₋₆alkyl    ester, C₁₋₅alkyl ester, and aryl ester), thiol, or thioether    substituents;

R⁹ is)N(R¹⁰ L-Z—R¹¹;

-   R¹⁰ is selected from hydrogen, OH, C₁₋₆aralkyl-Y—, and C₁₋₆alkyl-Y—,    preferably hydrogen;-   R¹¹ is selected from hydrogen, OR¹², C₁₋₆alkenyl, Ar—Y—,    carbocyclyl, and heterocyclyl; and-   R¹² is selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl,    preferably hydrogen.

In certain embodiments, L is selected from C═O, C═S, and SO₂, preferablySO₂ or C═O.

In certain embodiments, R¹⁰ is selected from hydrogen, OH, C₁₋₆aralkyl,and C₁₋₆alkyl, preferably hydrogen.

In certain embodiments, R¹¹ is selected from hydrogen, C₁₋₆alkenyl,Ar—Y—, carbocyclyl, and heterocyclyl.

In certain embodiments, R⁵ and R⁶ are each independently selected fromC₁₋₆alkyl, C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl. In preferred suchembodiments, R⁵ is C₁₋₆alkyl and R⁶ is C₁₋₆aralkyl. In more preferredsuch embodiments, R⁵ is isobutyl and R⁶ is phenylmethyl.

In certain embodiments, R¹⁰ is hydrogen, L is C═O or SO₂, R¹¹ is Ar—Y—,and each Ar is independently selected from phenyl, indolyl,benzofuranyl, naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl,pyrazyl, and the like. In certain such embodiments, Ar may besubstituted with Ar-Q-, where Q is selected from a direct bond, —O—, andC₁₋₆alkyl. In certain other such embodiments where Z is C₁₋₆alkyl, Z maybe substituted, preferably with Ar, e.g., phenyl.

In certain embodiments, R¹⁰ is hydrogen, Z is absent, L is C═O or SO₂,and R¹¹ is selected from Ar—Y and heterocyclyl. In certain preferredsuch embodiments, heterocyclyl is selected from chromonyl, chromanyl,morpholino, and piperidinyl. In certain other preferred suchembodiments, Ar is selected from phenyl, indolyl, benzofuranyl,naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl, pyrazyl, and thelike.

In certain embodiments, R¹⁰ is hydrogen, L is C═O or SO₂, Z is absent,and R¹¹ is C₁₋₆alkenyl, where C₁₋₆alkenyl is a substituted vinyl groupwhere the substituent is preferably an aryl or heteroaryl group, morepreferably a phenyl group optionally substituted with one to foursubstituents.

In certain embodiments, R¹² is selected from hydrogen and C₁₋₆alkyl. Incertain preferred such embodiments, R¹² is selected from hydrogen andmethyl. In more preferred such embodiments, R¹² is hydrogen.

In certain preferred embodiments, the chain of amino acids has astructure of formula (X)

X is COOH or an activated form thereof, preferably X is COOH, COCl, orCON(Me)(OMe), most preferably X is COOH or COCl;

R⁵, R⁶, and R⁷ are independently selected from C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether, preferably R⁶ is C₁₋₆alkyl and R⁵ and R⁷are C₁₋₆aralkyl, most preferably, R⁶ is isobutyl, R⁵ is 2-phenylethyl,and R⁷ is phenylmethyl;

R⁹ is a further chain of amino acids, hydrogen, C₁₋₆acyl, a protectinggroup, aryl, or heteroaryl, where substituents include halogen,carbonyl, nitro, hydroxy, aryl, and C₁₋₅alkyl, preferably R⁹ isC₁₋₆acyl, most preferably R⁹ is acetyl.

In certain preferred embodiments, the chain of amino acids has astructure of formula (XI) or a pharmaceutically acceptable salt thereof,

wherein

L is absent or is selected from —CO₂ or —C(═S)O;

X is COOH or an activated form thereof, preferably X is COOH, COCl, orCON(Me)(OMe), most preferably X is COOH or COCl;

Y is NH, N-alkyl, O, or C(R⁹)₂, preferably N-alkyl, O, or C(R⁹)₂;

Z is O or C(R⁹)₂, preferably C(R⁹)₂;

R¹, R², and R³ are independently selected from hydrogen and a group offormula (XII), preferably, R¹, R², and R³ are all the same, morepreferably R¹, R², and R³ are all hydrogen;

each R⁵, R⁶, R⁷, and R⁹ is independently selected from hydrogen,C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl,each of which is optionally substituted with a group selected fromalkyl, amide, amine, carboxylic acid or a pharmaceutically acceptablesalt thereof, carboxyl ester, thiol, and thioether, preferably R⁵, R⁶,and R⁷ are independently selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl, andC₁₋₆aralkyl and each R⁹ is hydrogen, more preferably, R⁶ is C₁₋₆alkyl,R⁵ and R⁷ are independently C₁₋₆aralkyl and each R⁹ is H;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₆alkyl, orR¹⁰ and R¹¹ together form a 3- to 6-membered carbocyclic or heterocyclicring;

R¹² and R¹³ are independently selected from hydrogen, a metal cation,C₁₋₆alkyl, and C₁₋₆aralkyl, or R¹² and R¹³ together represent C₁₋₆alkyl,thereby forming a ring;

m is an integer from 0 to 2; and

n is an integer from 0 to 2, preferably 0 or 1.

In certain embodiments, X is O and R¹, R², and R³ are all the same,preferably R¹, R², and R³ are all hydrogen. In certain such embodiments,R⁵, R⁶, and R⁷ are independently selected from C₁₋₆alkyl,C₁₋₆hydroxyalkyl, and C₁₋₆aralkyl, more preferably, R⁶ is C₁₋₆alkyl andR⁵ and R⁷ are independently C₁₋₆aralkyl.

In certain preferred embodiments, R¹, R², and R³ are all hydrogen, R⁶and R⁸ are both isobutyl, R⁵ is phenylethyl, and R⁷ is phenylmethyl.

In certain embodiments, R⁵, R⁶, and R⁷ are independently selected fromhydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, andC₁₋₆aralkyl, each of which is optionally substituted with a groupselected from alkyl, amide, amine, carboxylic acid or a pharmaceuticallyacceptable salt thereof, carboxyl ester, thiol, and thioether. Incertain embodiments, at least one of R⁵ and R⁷ is C₁₋₆aralkylsubstituted with alkyl, more preferably substituted with perhaloalkyl.In certain such embodiments, R⁷ is C₁₋₆aralkyl substituted withtrifluoromethyl.

In certain embodiments, Y is selected from N-alkyl, O, and CH₂. Incertain such embodiments, Z is CH₂, and m and n are both 0. In certainalternative such embodiments, Z is CH₂, m is 0, and n is 2 or 3. In yetanother alternative such embodiments, Z is O, m is 1, and n is 2.

In certain preferred embodiments, the chain of amino acids has astructure of formula (XIII)

wherein

each Ar is independently an aromatic or heteroaromatic group optionallysubstituted with 1 to 4 substituents;

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

B is absent or is N(R⁹)R¹⁰, preferably absent;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

X is COOH or an activated form thereof, preferably X is COOH, COCl, orCON(Me)(OMe), most preferably X is COOH or COCl;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R¹ is selected from H, —C₁₋₆alkyl-B, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl,aryl, and C₁₋₆aralkyl;

R² is selected from aryl, C₁₋₆aralkyl, heteroaryl, andC₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P (═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺-C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group, more preferablyt-butoxycarbonyl or benzyloxycarbonyl; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁷ is selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl, preferablyhydrogen;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

In certain embodiments, R¹ is selected from —C₁₋₆alkyl-B andC₁₋₆aralkyl. In certain such embodiments, R¹ is substituted with one ormore substituents selected from hydroxy, halogen, amide, amine,carboxylic acid (or a salt thereof), ester (including C₁₋₆alkyl ester,C₁₋₅alkylester, and aryl ester), thiol, or thioether. In certainpreferred such embodiments, R¹ is substituted with one or moresubstituents selected from carboxylic acid and ester. In certainembodiments, R¹ is selected from methyl, ethyl, isopropyl,carboxymethyl, and benzyl. In certain embodiments R¹ is —C₁₋₆alkyl-B andC₁₋₆aralkyl. In certain preferred such embodiments, B is absent.

In certain embodiments, R² is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain such embodiments, R² is selected fromC₁₋₆alkyl-phenyl, C₁₋₆alkyl-indolyl, C₁₋₆alkyl-thienyl,C₁₋₆alkyl-thiazolyl, and C₁₋₆alkyl-isothiazolyl, wherein the alkylmoiety may contain six, five, four, three, two, or one carbon atoms,preferably one or two. In certain such embodiments, R² is substitutedwith one or more substituents selected from hydroxy, halogen, amide,amine, carboxylic acid (or a salt thereof), ester (including C₁₋₆alkylester, C₁₋₅alkyl ester, and aryl ester), thiol, or thioether. In certainsuch embodiments, R² is substituted with a substituent selected fromalkyl, trihaloalkyl, alkoxy, hydroxy, or cyano. In certain suchembodiments, R² is selected from C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl.In certain preferred such embodiments, R² is selected from

-   -   R═H or any suitable protecting group        wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ and CH₃.        In certain embodiments D is selected from H, OMe, OH, CN, CF₃        and CH₃.

In certain preferred such embodiments where D is attached to asix-membered ring, D is attached at the 4-position relative to the pointof attachment, preferably excluding embodiments where the 4-position ofthe ring is occupied by the nitrogen of a pyridine ring.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, R⁶ is Ar—Y—,and each Ar is independently selected from phenyl, indolyl,benzofuranyl, naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl,pyrazyl, and the like. In certain such embodiments, Ar may besubstituted with Ar-E-, where E is selected from a direct bond, —O—, andC₁₋₆alkyl. In certain other such embodiments where Q is C₁₋₆alkyl, Q maybe substituted, preferably with Ar, e.g., phenyl.

In certain embodiments, R⁵ is hydrogen, Q is absent, L is C═O or SO₂,and R⁶ is selected from Ar—Y and heterocyclyl. In certain preferred suchembodiments, heterocyclyl is selected from chromonyl, chromanyl,morpholino, and piperidinyl. In certain other preferred suchembodiments, Ar is selected from phenyl, indolyl, benzofuranyl,naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl, pyrazyl, and thelike.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, Q is absent,and R⁶ is C₁₋₆alkenyl, where C₁₋₆alkenyl is a substituted vinyl groupwhere the substituent is preferably an aryl or heteroaryl group, morepreferably a phenyl group optionally substituted with one to foursubstituents.

In certain embodiments, L and Q are absent and R⁶ is selected fromC₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.In certain such embodiments, R⁵ is C₁₋₆alkyl and R⁶ is selected frombutyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R⁶ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R⁶ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R⁶ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹³)₂N—C₁₋₈alkyl-, (R¹³)₃N⁺-C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,R¹⁴SO₂C₁₋₈alkyl-, and R¹⁴SO₂NH—, wherein each occurrence of Z and A isindependently other than a covalent bond. In certain embodiments, L isC═O, Q is absent, and R⁶ is H.

In certain embodiments, R⁵ is C₁₋₆alkyl, R⁶ is C₁₋₆alkyl, Q is absent,and L is C═O. In certain such embodiments, R⁶ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R⁶ is C₁₋₆aralkyl. Incertain such embodiments, R⁶ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R⁵ is C₁₋₆alkyl, and R⁶ isaryl. In certain such embodiments, R⁶ is substituted or unsubstitutedphenyl.

In certain embodiments, L is C═O, Q is absent, and R⁶ is selected fromheteroaryl and C₁₋₆heteroaralkyl. In certain such embodiments, R⁶ isheteroaryl selected from pyrrole, furan, thiophene, imidazole,isoxazole, oxazole, oxadiazole, thiazole, thiadiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. In certainalternative such embodiments, R⁶ is C₁₋₆heteroaralkyl selected frompyrrolylmethyl, furanylmethyl, thienylmethyl, imidazolylmethyl,isoxazolylmethyl, oxazolylmethyl, oxadiazolylmethyl, thiazolylmethyl,thiadiazolylmethyl, triazolylmethyl, pyrazolylmethyl, pyridylmethyl,pyrazinylmethyl, pyridazinylmethyl and pyrimidinylmethyl.

In certain embodiments, L is C═O, Q is absent or O, and R⁶ iscarbocyclylM-, wherein M is C₀₋₁alkyl. In certain such embodiments, R⁶is cyclopropyl or cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, M isC₁₋₈alkyl, preferably methylene, and R⁶ is selected fromR¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-, R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-, wherein each occurrence of A isindependently other than a covalent bond. In certain such embodiments,R⁶ is heterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R¹⁶)(R¹⁷), wherein R¹⁶ and R¹⁷ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, preferably C₁₋₃alkyl-Y—C₁₋₃alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, M is C₁₋₈alkyl,and R⁶ is selected from (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂NC₁₋₈alkyl,(R¹³)₃N⁺C₁₋₈alkyl-, and heterocyclyl-M-. In certain such embodiments, R⁶is (R¹³)₂NC₁₋₈alkyl or (R¹³)₃N′C₁₋₈alkyl-, where R¹³ is C₁₋₆alkyl. Incertain other such embodiments, R⁶ is heterocyclylM-, where heterocyclylis selected from morpholino, piperidino, piperazino, and pyrrolidino.

In certain embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q is selected from Oand NH and R⁶ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl. In other embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q isselected from O and NH, and R⁶ is C₁₋₆alkyl, where C₁₋₆alkyl is selectedfrom methyl, ethyl, and isopropyl. In further embodiments, L is C═O, R⁵is C₁₋₆alkyl, Q is selected from O and NH and R⁶ is C₁₋₆aralkyl, wherearalkyl is phenylmethyl. In other embodiments, L is C═O, R⁵ isC₁₋₆alkyl, Q is selected from O and NH, and R⁶ is C₁₋₆heteroaralkyl,where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R⁵ and R⁶ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,wherein each occurrence of Z and A is independently other than acovalent bond, thereby forming a ring. In certain preferred embodiments,L is C═O, Q and Y are absent, and R⁵ and R⁶ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and Q areabsent, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In anotherpreferred embodiment, L is C═O, Q is absent, Y is selected from NH andN—C₁₋₆alkyl, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L is C═O, Y is absent, and R⁵ and R⁶together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Land A are C═O, and R⁵ and R⁶ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. Inanother preferred embodiment, L and A are C═O and R⁵ and R⁶ together areC₂₋₃alkyl-A.

In certain embodiments, R⁷ is selected from hydrogen and C₁₋₆alkyl. Incertain preferred such embodiments, R⁷ is selected from hydrogen andmethyl. In more preferred such embodiments, R⁷ is hydrogen.

In certain embodiments, R² and R³ are each independently C₁₋₆aralkyl,and R¹ is selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl,aryl, and C₁₋₆aralkyl, any of which is optionally substituted with oneor more of amide, amine, carboxylic acid (or a salt thereof), ester(including C₁₋₆alkyl ester, C₁₋₅alkyl ester, and aryl ester), thiol, orthioether substituents.

In certain preferred embodiments, the chain of amino acids has astructure of formula (XIV)

each Ar is independently an aromatic or heteroaromatic group optionallysubstituted with 1 to 4 substituents;

each A is independently selected from C═O, C═S, and SO₂, preferably C═O;or

A is optionally a covalent bond when adjacent to an occurrence of Z;

L is absent or is selected from C═O, C═S, and SO₂, preferably SO₂ orC═O;

M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;

Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;

X is COOH or an activated form thereof, preferably X is COOH, COCl, orCON(Me)(OMe), most preferably X is COOH or COCl;

Y is absent or is selected from C═O and SO₂;

each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,preferably O; or

Z is optionally a covalent bond when adjacent to an occurrence of A;

R² is selected from aryl, C₁₋₆aralkyl, heteroaryl, andC₁₋₆heteroaralkyl;

R⁴ is N(R⁵)L-Q-R⁶;

R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl, preferablyhydrogen;

R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group, aryl,C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R¹¹ZAZ—C₁₋₈alkyl-,R¹⁴Z—C₁₋₈alkyl-, (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,(R¹³)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-,and R¹⁴SO₂NH; preferably an N-capping group, more preferablyt-butoxycarbonyl or benzyloxycarbonyl; or

R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, therebyforming a ring;

R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably C₁₋₆alkyl;and

R¹⁰ is an N-terminal protecting group;

R¹¹ and R¹² are independently selected from hydrogen, metal cation,C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, andC₁₋₆alkyl, or R¹¹ and R¹² together are C₁₋₆alkyl, thereby forming aring;

each R¹³ is independently selected from hydrogen and C₁₋₆alkyl,preferably C₁₋₆alkyl; and

R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C₁₋₆aralkyl,and C₁₋₆heteroaralkyl;

R¹⁵ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxy,—C(O)OC₁₋₆alkyl, —C(O)NHC₁₋₆alkyl, and C₁₋₆aralkyl, preferably C₁₋₆alkyland C₁₋₆hydroxyalkyl, more preferably methyl, ethyl, hydroxymethyl, and2-hydroxyethyl;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

In certain embodiments, R² is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain such embodiments, R² is selected fromC₁₋₆alkyl-phenyl, C₁₋₆alkyl-indolyl, C₁₋₆alkyl-thienyl,C₁₋₆alkyl-thiazolyl, and C₁₋₆alkyl-isothiazolyl, wherein the alkylmoiety may contain six, five, four, three, two, or one carbon atoms,preferably one or two. In certain such embodiments, R² is substitutedwith one or more substituents selected from hydroxy, halogen, amide,amine, carboxylic acid (or a salt thereof), ester (including C₁₋₆alkylester, C₁₋₅alkyl ester, and aryl ester), thiol, or thioether. In certainsuch embodiments, R² is substituted with a substituent selected fromalkyl, trihaloalkyl, alkoxy, hydroxy, or cyano. In certain suchembodiments, R² is selected from C₁₋₆alkyl-phenyl and C₁₋₆alkyl-indolyl.In certain preferred such embodiments, R² is selected from

-   -   R═H or any suitable protecting group        wherein D is selected from H, OMe, OBu^(t), OH, CN, CF₃ and CH₃.        In certain embodiments D is selected from H, OMe, OH, CN, CF₃        and CH₃.

In certain preferred such embodiments where D is attached to asix-membered ring, D is attached at the 4-position relative to the pointof attachment, preferably excluding embodiments where the 4-position ofthe ring is occupied by the nitrogen of a pyridine ring.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, R⁶ is Ar—Y—,and each Ar is independently selected from phenyl, indolyl,benzofuranyl, naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl,pyrazyl, and the like. In certain such embodiments, Ar may besubstituted with Ar-E-, where E is selected from a direct bond, —O—, andC₁₋₆alkyl. In certain other such embodiments where Q is C₁₋₆alkyl, Q maybe substituted, preferably with Ar, e.g., phenyl.

In certain embodiments, R⁵ is hydrogen, Q is absent, L is C═O or SO₂,and R⁶ is selected from Ar—Y and heterocyclyl. In certain preferred suchembodiments, heterocyclyl is selected from chromonyl, chromanyl,morpholino, and piperidinyl. In certain other preferred suchembodiments, Ar is selected from phenyl, indolyl, benzofuranyl,naphthyl, quinolinyl, quinolonyl, thienyl, pyridyl, pyrazyl, and thelike.

In certain embodiments, R⁵ is hydrogen, L is C═O or SO₂, Q is absent,and R⁶ is C₁₋₆alkenyl, where C₁₋₆alkenyl is a substituted vinyl groupwhere the substituent is preferably an aryl or heteroaryl group, morepreferably a phenyl group optionally substituted with one to foursubstituents.

In certain embodiments, L and Q are absent and R⁶ is selected fromC₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.In certain such embodiments, R⁵ is C₁₋₆alkyl and R⁶ is selected frombutyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R⁶ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R⁶ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R⁶ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,(R¹³)₂N—C₁₋₈alkyl-, (R¹³)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,R¹⁴SO₂C₁₋₈alkyl-, and R¹⁴SO₂NH—, wherein each occurrence of Z and A isindependently other than a covalent bond. In certain embodiments, L isC═O, Q is absent, and R⁶ is H.

In certain embodiments, R⁵ is C₁₋₆alkyl, R⁶ is C₁₋₆alkyl, Q is absent,and L is C═O. In certain such embodiments, R⁶ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R⁶ is C₁₋₆aralkyl. Incertain such embodiments, R⁶ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R⁵ is C₁₋₆alkyl, and R⁶ isaryl. In certain such embodiments, R⁶ is substituted or unsubstitutedphenyl.

In certain embodiments, L is C═O, Q is absent, and R⁶ is selected fromheteroaryl and C₁₋₆heteroaralkyl. In certain such embodiments, R⁶ isheteroaryl selected from pyrrole, furan, thiophene, imidazole,isoxazole, oxazole, oxadiazole, thiazole, thiadiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. In certainalternative such embodiments, R⁶ is C₁₋₆heteroaralkyl selected frompyrrolylmethyl, furanylmethyl, thienylmethyl, imidazolylmethyl,isoxazolylmethyl, oxazolylmethyl, oxadiazolylmethyl, thiazolylmethyl,thiadiazolylmethyl, triazolylmethyl, pyrazolylmethyl, pyridylmethyl,pyrazinylmethyl, pyridazinylmethyl and pyrimidinylmethyl.

In certain embodiments, L is C═O, Q is absent or O, and R⁶ iscarbocyclylM-, wherein M is C₀₋₁alkyl. In certain such embodiments, R⁶is cyclopropyl or cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, M isC₁₋₈alkyl, preferably methylene, and R⁶ is selected fromR¹¹ZA-C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-, R¹¹ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-, wherein each occurrence of A isindependently other than a covalent bond. In certain such embodiments,R⁶ is heterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R¹⁶)(R¹⁷) wherein R¹⁶ and R¹⁷ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, preferably C₁₋₃alkyl-Y—C₁₋₃alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, M is C₁₋₈alkyl,and R⁶ is selected from (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂NC₁₋₈alkyl,(R¹³)₃N⁺C₁₋₈alkyl-, and heterocyclyl-M-. In certain such embodiments, R⁶is (R¹³)₂NC₁₋₈alkyl or (R¹³)₃N⁺C₁₋₈alkyl-, where R¹³ is C₁₋₆alkyl. Incertain other such embodiments, R⁶ is heterocyclylM-, where heterocyclylis selected from morpholino, piperidino, piperazino, and pyrrolidino.

In certain embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q is selected from Oand NH and R⁶ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl. In other embodiments, L is C═O, R⁵ is C₁₋₆alkyl, Q isselected from O and NH, and R⁶ is C₁₋₆alkyl, where C₁₋₆alkyl is selectedfrom methyl, ethyl, and isopropyl. In further embodiments, L is C═O, R⁵is C₁₋₆alkyl, Q is selected from O and NH and R⁶ is C₁₋₆aralkyl, wherearalkyl is phenylmethyl. In other embodiments, L is C═O, R⁵ isC₁₋₆alkyl, Q is selected from O and NH, and R⁶ is C₁₋₆heteroaralkyl,where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R⁵ and R⁶ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,wherein each occurrence of Z and A is independently other than acovalent bond, thereby forming a ring. In certain preferred embodiments,L is C═O, Q and Y are absent, and R⁵ and R⁶ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and Q areabsent, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In anotherpreferred embodiment, L is C═O, Q is absent, Y is selected from NH andN—C₁₋₆alkyl, and R⁵ and R⁶ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L is C═O, Y is absent, and R⁵ and R⁶together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Land A are C═O, and R⁵ and R⁶ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. Inanother preferred embodiment, L and A are C═O and R⁵ and R⁶ together areC₂₋₃alkyl-A.

In certain embodiments, R² is C₁₋₆aralkyl, and R¹ is selected fromC₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, anyof which is optionally substituted with one or more of amide, amine,carboxylic acid (or a salt thereof), ester (including C₁₋₆alkyl ester,C₁₋₅alkyl ester, and aryl ester), thiol, or thioether substituents.

In certain preferred embodiments, the chain of amino acids has astructure of formula (XV)

wherein

L is selected from C═O, C═S, and SO₂, preferably C═O;

X is COOH or an activated form thereof, preferably X is COOH, COCl, orCON(Me)(OMe), most preferably X is COOH or COCl;

Z is absent, C₁₋₆alkyl, C₁₋₆alkoxy, or NR, e.g., absent, C₁₋₆alkyl, orC₁₋₆alkoxy, preferably absent;

R is selected from H and C₁₋₆alkyl, preferably H or CH₃;

R¹ and R² are each independently selected from hydrogen, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl,C₁₋₆aralkyl, heteroaryl, heterocyclyl, C₁₋₆heterocycloalkyl,C₁₋₆heteroaralkyl, carbocyclyl, and C₁₋₆-carbocyclolalkyl;

R⁴ is selected from hydrogen, C₁₋₆aralkyl, and C₁₋₆alkyl;

R⁵ is heteroaryl; and

R⁶ is selected from hydrogen, C₁₋₆alkyl, and C₁₋₆aralkyl.

In certain embodiments, R¹ and R² are independently selected fromhydrogen, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, C₁₋₆heteroaralkyl, and C₁₋₆-carbocyclolalkyl. Incertain embodiments, R¹ and R² are independently C₁₋₆alkyl selected frommethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and isobutyl. Incertain embodiments, R¹ and R² are independently C₁₋₆hydroxyalkyl. Incertain preferred such embodiments, R¹ and R² are independently selectedfrom hydroxymethyl and hydroxyethyl, preferably hydroxymethyl. Incertain embodiments, R¹ and R² are independently C₁₋₆alkoxyalkyl. Incertain such embodiments, R¹ and R² are independently selected frommethoxymethyl and methoxyethyl, preferably methoxymethyl. In certainembodiments, R¹ and R² are independently C₁₋₆heteroaralkyl. In certainsuch embodiments, R¹ and R² are independently selected fromimidazolylmethyl, pyrazolylmethyl, and thiazolylmethyl, andpyridylmethyl, preferably imidazol-4-ylmethyl, thiazol-4-ylmethyl,2-pyridylmethyl, 3-pyridylmethyl, or 4-pyridylmethyl. In certainembodiments, R¹ and R² are independently C₁₋₆aralkyl. In certain suchembodiments, R¹ and R² are independently selected from phenylmethyl(benzyl) and phenylethyl, preferably phenylmethyl. In certainembodiments, R¹ and R² are independently C₁₋₆-carbocycloalkyl. Incertain such embodiments R¹ is cyclohexylmethyl. In certain embodimentsR¹ and R² are different. In certain embodiments, R¹ and R² are the same.

In certain embodiments, at least one of R¹ and R² is selected fromC₁₋₆hydroxyalkyl and C₁₋₆alkoxyalkyl. In certain such embodiments, atleast one of R¹ and R² is alkoxyalkyl. In certain such embodiments, atleast one of R¹ and R² is selected from methoxymethyl and methoxyethyl.

In certain embodiments, R⁴ and R⁶ are independently selected fromhydrogen and methyl, preferably hydrogen.

In certain embodiments, R⁵ is a 5- or 6-membered heteroaryl. In certainsuch embodiments, R⁵ is selected from isoxazole, isothiazole, furan,thiophene, oxazole, thiazole, pyrazole, or imidazole, preferablyisoxazole, furan, or thiazole.

In certain embodiments, R⁵ is a bicyclic heteroaryl. In certain suchembodiments bicyclic heteroaryl is selected from benzisoxazole,benzoxazole, benzothiazole, benzisothiazole.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a1,3-thiazol-5-yl or 1,3-thiazol-4-yl. In certain such embodiments, whenthe thiazole is substituted, it is substituted at least at the2-position. In other such embodiments, R⁵ is an unsubstituted1,3-thiazol-5-yl or 1,3-thiazol-4-yl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a substituted1,3-thiazol-5-yl. In certain such embodiments, R⁵ is 1,3-thiazol-5-ylsubstituted with a substituent selected from C₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyalkyl, C₁₋₆hydroxyalkyl, carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂-aminocarboxylate,C₁₋₆alkylcarboxylate, C₁₋₆heteroaralkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, and C₁₋₆-carbocycloalkyl. In certain preferredsuch embodiments, R⁵ is 1,3-thiazol-5-yl substituted with a substituentselected from methyl, ethyl, isopropyl, and cyclopropylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a substituted1,3-thiazol-4-yl. In certain such embodiments, R⁵ is 1,3-thiazol-4-ylsubstituted with a substituent selected from C₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyalkyl, C₁₋₆hydroxyalkyl, carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate,C₁₋6alkylcarboxylate, C₁₋₆heteroaralkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, and C₁₋₆carbocycloalkyl. In certain preferred suchembodiments, R⁵ is 1,3-thiazol-4-yl substituted with a substituentselected from methyl, ethyl, isopropyl, and cyclopropylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anisoxazol-3-yl or isoxazol-5-yl. In certain preferred such embodiments,when the isoxazol-3-yl is substituted, it is substituted at least at the5-position. In certain preferred embodiments, when the isoxazol-5-yl issubstituted, it is substituted at least at the 3-position.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anunsubstituted isoxazol-3-yl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a substitutedisoxazol-3-yl. In certain such embodiments, R⁵ is isoxazol-3-ylsubstituted with a substituent selected from C₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyalkyl, C₁₋₆hydroxyalkyl, carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate,C₁₋₆alkylcarboxylate, C₁₋₆heteroaralkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, and C₁₋₆carbocycloalkyl. In certain preferred suchembodiments R⁵ is isoxazole-3-yl substituted with a substituent selectedfrom methyl, ethyl, isopropyl, and cyclopropylmethyl.

In certain embodiments L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a 4- to 6-membered nitrogen-containingC₁₋₆heterocycloalkyl. In certain such embodiments, R⁵ is isoxazol-3-ylsubstituted with azetidinylmethyl, preferably azetidin-1-ylmethyl. Incertain alternative such embodiments, L is C═O, Z is absent, and R⁵ isisoxazol-3-yl substituted with

wherein W is O, NR, or CH₂, and R is H or C₁₋₆alkyl. In certain suchembodiments, W is O.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with 5-membered nitrogen-containing C₁₋₆heteroaralkyl, suchas pyrazolylmethyl, imidazolylmethyl, triazol-5-ylmethyl, preferably1,2,4-triazol-5-ylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with C₁₋₆alkoxy or C₁₋₆alkoxyalkyl, preferably methoxy,ethoxy, methoxymethyl, or methoxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with C₁₋₆hydroxyalkyl, preferably hydroxymethyl orhydroxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-3-ylsubstituted with a carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate, orC₁₋₆alkylcarboxylate. In certain such embodiments, R⁵ is substitutedwith methyl carboxylate or ethyl carboxylate, preferably methylcarboxylate.

In certain embodiments, L is C═O, Z is absent, and R⁵ is anunsubstituted isoxazol-5-yl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is a substitutedisoxazol-5-yl. In certain such embodiments, R⁵ is isoxazol-5-ylsubstituted with a substituent selected from C₁₋₆alkyl, C₁₋₆alkoxy,C₁₋₆alkoxyalkyl, C₁₋₆hydroxyalkyl, carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate,C₁₋₆alkylcarboxylate, C₁₋₆heteroaralkyl, C₁₋₆aralkyl,C₁₋₆heterocycloalkyl, and C₁₋₆carbocycloalkyl In certain preferred suchembodiments R⁵ is isoxazole-5-yl substituted with a substituent selectedfrom methyl, ethyl, isopropyl, and cyclopropylmethyl.

In certain embodiments L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with a 4- to 6-membered nitrogen-containingC₁₋₆heterocycloalkyl. In certain such embodiments, R⁵ is isoxazol-5-ylsubstituted with azetidinylmethyl, preferably azetidin-1-ylmethyl. Incertain alternative such embodiments, L is C═O, Z is absent, and R⁵ isisoxazol-5-yl substituted with

wherein W is O, NR, or CH₂, and R is H or C₁₋₆alkyl. In certain suchembodiments, W is O.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with 5-membered nitrogen-containing C₁₋₆heteroaralkyl, suchas pyrazolylmethyl, imidazolylmethyl, triazol-5-ylmethyl, preferably1,2,4-triazol-5-ylmethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with C₁₋₆alkoxy or C₁₋₆alkoxyalkyl, preferably methoxy,ethoxy, methoxymethyl, or methoxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with C₁₋₆hydroxyalkyl, preferably hydroxymethyl orhydroxyethyl.

In certain embodiments, L is C═O, Z is absent, and R⁵ is isoxazol-5-ylsubstituted with a carboxylic acid, aminocarboxylate,C₁₋₆alkylaminocarboxylate, (C₁₋₆alkyl)₂aminocarboxylate, orC₁₋₆alkylcarboxylate. In certain such embodiments, R⁵ is substitutedwith methyl carboxylate or ethyl carboxylate, preferably methylcarboxylate.

In certain embodiments, Z is NR, preferably NH.

Uses of Enzyme Inhibitors

Orderly protein degradation is crucial to the maintenance of normal cellfunctions, and the proteasome is integral to the protein degradationprocess. The proteasome controls the levels of proteins that areimportant for cell-cycle progression and apoptosis in normal andmalignant cells; for example, cyclins, caspases, BCL2 and nF-kB(Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075; Almondet al., Leukemia (2002) 16: 433-443). Thus, it is not surprising thatinhibiting proteasome activity can translate into therapies to treatvarious disease states, such as malignant, non-malignant and autoimmunediseases, depending on the cells involved.

Both in vitro and in vivo models have shown that malignant cells, ingeneral, are susceptible to proteasome inhibition. In fact, proteasomeinhibition has already been validated as a therapeutic strategy for thetreatment of multiple myeloma. This could be due, in part, to the highlyproliferative malignant cell's dependency on the proteasome system torapidly remove proteins (Rolfe et al., J. Mol. Med. (1997) 75:5-17;Adams, Nature (2004) 4: 349-360). Therefore, certain embodiments of theinvention relate to a method of treating cancers comprisingadministering to a subject in need of such treatment an effective amountof the proteasome inhibitor compound disclosed herein. As used herein,the term “cancer” includes, but is not limited to, blood born and solidtumors. Cancer refers to disease of blood, bone, organs, skin tissue andthe vascular system, including, but not limited to, cancers of thebladder, blood, bone, brain, breast, cervix, chest, colon, endometrium,esophagus, eye, head, kidney, liver, lung, lymph nodes, mouth, neck,ovaries, pancreas, prostate, rectum, renal, skin, stomach, testis,throat, and uterus. Specific cancers include, but are not limited to,leukemia (acute lymphocytic leukemia (ALL), acute lyelogenous leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia(CML), hairy cell leukemia), mature B cell neoplasms (small lymphocyticlymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma(such as Waldenström's macroglobulinemia), splenic marginal zonelymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulindeposition diseases, heavy chain diseases, extranodal marginal zone Bcell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma(NMZL), follicular lymphoma, mantle cell lymphoma, diffuse B celllymphoma, mediastinal (thymic) large B cell lymphoma, intravascularlarge B cell lymphoma, primary effusion lymphoma and Burkittlymphoma/leukemia), mature T cell and natural killer (NK) cell neoplasms(T cell prolymphocytic leukemia, T cell large granular lymphocyticleukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma,extranodal NK/T cell lymphoma, enteropathy-type T cell lymphoma,hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosisfungoides (Sezary syndrome), primary cutaneous anaplastic large celllymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma,unspecified peripheral T cell lymphoma and anaplastic large celllymphoma), Hodgkin lymphoma (nodular sclerosis, mixed celluarity,lymphocyte-rich, lymphocyte depleted or not depleted, nodularlymphocyte-predominant), myeloma (multiple myeloma, indolent myeloma,smoldering myeloma), chronic myeloproliferative disease,myelodysplastic/myeloproliferative disease, myelodysplastic syndromes,immunodeficiency-associated lymphoproliferative disorders, histiocyticand dendritic cell neoplasms, mastocytosis, chondrosarcoma, Ewingsarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bone disease,osteosarcoma, breast cancer (hormone dependent, hormone independent),gynecological cancers (cervical, endometrial, fallopian tube,gestational trophoblastic disease, ovarian, peritoneal, uterine, vaginaland vulvar), basal cell carcinoma (BCC), squamous cell carcinoma (SCC),malignant melanoma, dermatofibrosarcoma protuberans, Merkel cellcarcinoma, Kaposi's sarcoma, astrocytoma, pilocytic astrocytoma,dysembryoplastic neuroepithelial tumor, oligodendrogliomas, ependymoma,glioblastoma multiforme, mixed gliomas, oligoastrocytomas,medulloblastoma, retinoblastoma, neuroblastoma, germinoma, teratoma,malignant mesothelioma (peritoneal mesothelioma, pericardialmesothelioma, pleural mesothelioma), gastro-entero-pancreatic orgastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid,pancreatic endocrine tumor (PET), colorectal adenocarcinoma, colorectalcarcinoma, aggressive neuroendocrine tumor, leiomyosarcomamucinousadenocarcinoma, Signet Ring cell adenocarcinoma, hepatocellularcarcinoma, cholangiocarcinoma, hepatoblastoma, hemangioma, hepaticadenoma, focal nodular hyperplasia (nodular regenerative hyperplasia,hamartoma), non-small cell lung carcinoma (NSCLC) (squamous cell lungcarcinoma, adenocarcinoma, large cell lung carcinoma), small cell lungcarcinoma, thyroid carcinoma, prostate cancer (hormone refractory,androgen independent, androgen dependent, hormone-insensitive), and softtissue sarcomas (fibrosarcoma, malignant fibrous hystiocytoma,dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma leiomyosarcoma,hemangiosarcoma, synovial sarcoma, malignant peripheral nerve sheathtumor/neurofibrosarcoma, extraskeletal osteosarcoma).

Many tumors of the haematopoietic and lymphoid tissues are characterizedby an increase in cell proliferation, or a particular type of cell. Thechronic myeloproliferative diseases (CMPDs) are clonal haematopoieticstem cell disorders characterized by proliferation in the bone marrow ofone or more of the myeloid lineages, resulting in increased numbers ofgranulocytes, red blood cells and/or platelets in the peripheral blood.As such, the use of proteasome inhibitors for the treatment of suchdiseases is attractive and being examined (Cilloni et al., Haematologica(2007) 92: 1124-1229). CMPD can include chronic myelogenous leukaemia,chronic neutrophilic leukaemia, chronic eosinophilic leukaemia,polycythaemia vera, chronic idiopathic myelofibrosis, essentialthrombocythaemia and unclassifiable chronic myeloproliferative disease.An aspect of the invention is the method of treating CMPD comprisingadministering to a subject in need of such treatment an effective amountof the proteasome inhibitor compound disclosed herein.

Myelodisplastic/myeloproliferative diseases, such as chronicmyelomonocytic leukaemia, atypical chronic myeloid leukemia, juvenilemyelomonocytic leukaemia and unclassifiablemyelodysplastic/myeloproliferative disease, are characterized byhypercellularity of the bone marrow due to proliferation in one or moreof the myeloid lineages. Inhibiting the proteasome with the compositiondescribed herein, can serve to treat thesemyelodisplatic/myeloproliferative diseases by providing a subject inneed of such treatment an effective amount or the composition.

Myelodysplastic syndromes (MDS) refer to a group of hematopoietic stemcell disorders characterized by dysplasia and ineffective haematopoiesisin one or more of the major myeloid cell lines. Targeting NF-kB with aproteasome inhibitor in these hematologic malignancies inducesapoptosis, thereby killing the malignant cell (Braun et al. Cell Deathand Differentiation (2006) 13:748-758). A further embodiment of theinvention is a method to treat MDS comprising administering to a subjectin need of such treatment an effective amount of the compound disclosedherein. MDS includes refractory anemia, refractory anemia with ringedsideroblasts, refractory cytopenia with multilineage dysplasia,refractory anemia with excess blasts, unclassifiable myelodysplasticsyndrome and myelodysplastic syndrome associated with isolated del(5q)chromosome abnormality.

Mastocytosis is a proliferation of mast cells and their subsequentaccumulation in one or more organ systems. Mastocytosis includes, but isnot limited to, cutaneous mastocytosis, indolent systemic mastocytosis(ISM), systemic mastocytosis with associated clonal haematologicalnon-mast-cell-lineage disease (SM-AHNMD), aggressive systemicmastocytosis (ASM), mast cell leukemia (MCL), mast cell sarcoma (MCS)and extrcutaneous mastocytoma. Another embodiment of the invention is amethod to treat mastocytosis comprising administering an effect amountof the compound disclosed herein to a subject diagnosed withmastocytosis.

The proteasome regulates NF-κB, which in turn regulates genes involvedin the immune and inflammatory response. For example, NF-κB is requiredfor the expression of the immunoglobulin light chain κ gene, the IL-2receptor α-chain gene, the class I major histocompatibility complexgene, and a number of cytokine genes encoding, for example, IL-2, IL-6,granulocyte colony-stimulating factor, and IFN-β (Palombella et al.,Cell (1994) 78:773-785). Thus, in certain embodiments, the inventionrelates to methods of affecting the level of expression of IL-2, MHC-I,IL-6, TNFα, IFN-β or any of the other previously-mentioned proteins,each method comprising administering to a subject an effective amount ofa proteasome inhibitor composition disclosed herein. In certainembodiments, the invention includes a method of treating an autoimmunedisease in a mammal comprising administering a therapeutically effectiveamount of the compound described herein. An “autoimmune disease” hereinis a disease or disorder arising from and directed against anindividual's own tissues. Examples of autoimmune diseases or disordersinclude, but are not limited to, inflammatory responses such asinflammatory skin diseases including psoriasis and dermatitis (e.g.atopic dermatitis); systemic scleroderma and sclerosis; responsesassociated with inflammatory bowel disease (such as Crohn's disease andulcerative colitis); respiratory distress syndrome (including adultrespiratory distress syndrome; ARDS); dermatitis; meningitis;encephalitis; uveitis; colitis; glomerulonephritis; allergic conditionssuch as eczema and asthma and other conditions involving infiltration ofT cells and chronic inflammatory responses; atherosclerosis; leukocyteadhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus(SLE); diabetes mellitus (e.g. Type I diabetes mellitus or insulindependent diabetes mellitis); multiple sclerosis; Reynaud's syndrome;autoimmune thyroiditis; allergic encephalomyelitis; Sjorgen's syndrome;juvenile onset diabetes; and immune responses associated with acute anddelayed hypersensitivity mediated by cytokines and T-lymphocytestypically found in tuberculosis, sarcoidosis, polymyositis,granulomatosis and vasculitis; pernicious anemia (Addison's disease);diseases involving leukocyte diapedesis; central nervous system (CNS)inflammatory disorder; multiple organ injury syndrome; hemolytic anemia(including, but not limited to cryoglobinemia or Coombs positiveanemia); myasthenia gravis; antigen-antibody complex mediated diseases;anti-glomerular basement membrane disease; antiphospholipid syndrome;allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome;pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; Reiter'sdisease; stiff-man syndrome; Beheet disease; giant cell arteritis;immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

The immune system screens for autologous cells that are virallyinfected, have undergone oncogenic transformation or present unfamiliarpeptides on their surface. Intracellular proteolysis generate smallpeptides for presentation to T-lymphocytes to induce MHC classI-mediated immune responses. Thus, in certain embodiments, the inventionrelates to a method of using the compound as an immunomodulatory agentfor inhibiting or altering antigen presentation in a cell, comprisingexposing the cell (or administering to a subject) to the compounddescribed herein. Specific embodiments include a method of treatinggraft or transplant-related diseases, such as graft-versus-host diseaseor host versus-graft disease in a mammal, comprising administering atherapeutically effective amount of the compound described herein. Theterm “graft” as used herein refers to biological material derived from adonor for transplantation into a recipient. Grafts include such diversematerial as, for example, isolated cells such as islet cells; tissuesuch as the amniotic membrane of a newborn, bone marrow, hematopoieticprecursor cells, and ocular tissue, such as corneal tissue; and organssuch as skin, heart. liver, spleen, pancreas, thyroid lobe. lung,kidney, tubular organs (e.g., intestine, blood vessels, or esophagus).The tubular organs can be used to replace damaged portions of esophagus,blood vessels, or bile duct. The skin grafts can be used not only forburns, but also as a dressing to damaged intestine or to close certaindefects such as diaphragmatic hernia. The graft is derived from anymammalian source, including human, whether from cadavers or livingdonors. In some cases, the donor and recipient is the same mammal.Preferably the graft is bone marrow or an organ such as heart and thedonor of the graft and the host are matched for HLA class II antigens.

Histiocytic and dendritic cell neoplasms are derived from phagocytes andaccessory cells, which have major roles in the processing andpresentation of antigens to lymphocytes. Depleting the proteasomecontent in dendritic cells has been shown to alter their antigen-inducedresponses (Chapatte et al. Cancer Res. (2006) 66:5461-5468). Thus,another embodiment of the invention comprises administering an effectiveamount of the composition disclosed herein to a subject with histiocyticor dendritic cell neoplasm. Histiocytic and dindritirc cell neoplasmsinclude histiocytic sarcoma, Langerhans cell histiocytosis, Langerhanscell sarcoma, interdigitating dendritic cell sarcoma/tumor, folliculardendritic cell sarcoma/tumor and non-specified dendritic cell sarcoma.

Inhibition of the proteasome has been shown to be beneficial to treatdiseases whereby a cell type is proliferating and immune disorders;thus, an embodiment of the invention includes the treatment oflymphoproliferative diseases (LPD) associated with primary immunedisorders (PID) comprising administering an effective amount of thedisclosed compound to a subject in need thereof. The most commonclinical settings of immunodeficiency associated with an increasedincidence of lymphoproliferative disorders, including B-cell and T-cellneoplasms and lymphomas, are primary immunodeficiency syndromes andother primary immune disorders, infection with the humanimmunodeficiency virus (HIV), iatrogenic immunosuppression in patientswho have received solid organ or bone marrow allografts, and iatrogenisimmunosuppression associated with methotrexate treatment. Other PIDscommonly associated with LPDs, but not limited to, are ataxiatelangiectasia (AT), Wiskott-Aldrich syndrome (WAS), common variableimmunodeficiency (CVID), severe combined immunodeficiency (SCID),X-linked lymphoproliferative disorder (XLP), Nijmegen breakage syndrome(NBS), hyper-IgM syndrome, and autoimmune lymphoproliferative syndrome(ALPS).

Additional embodiments of the invention relate to methods for affectingthe proteasome-dependent regulation of oncoproteins and methods oftreating or inhibiting cancer growth, each method comprising exposing acell (in vivo, e.g., in a subject, or in vitro) to the proteasomeinhibitor composition disclosed herein. HPV-16 and HPV-18-derived E6proteins stimulate ATP- and ubiquitin-dependent conjugation anddegradation of p53 in crude reticulocyte lysates. The recessive oncogenep53 has been shown to accumulate at the nonpermissive temperature in acell line with a mutated thermolabile E1. Elevated levels of p53 maylead to apoptosis. Examples of proto-oncoproteins degraded by theubiquitin system include c-Mos, c-Fos, and c-Jun. In certainembodiments, the invention relates to a method for treating p53-relatedapoptosis, comprising administering to a subject an effective amount ofa proteasome inhibitor composition disclosed herein.

Another aspect of the invention relates to the use of proteasomeinhibitor compositions disclosed herein for the treatment ofneurodegenerative diseases and conditions, including, but not limitedto, stroke, ischemic damage to the nervous system, neural trauma (e.g.,percussive brain damage, spinal cord injury, and traumatic damage to thenervous system), multiple sclerosis and other immune-mediatedneuropathies (e.g., Guillain-Bane syndrome and its variants, acute motoraxonal neuropathy, acute inflammatory demyelinating polyneuropathy, andFisher Syndrome), HIV/AIDS dementia complex, axonomy, diabeticneuropathy, Parkinson's disease, Huntington's disease, multiplesclerosis, bacterial, parasitic, fungal, and viral meningitis,encephalitis, vascular dementia, multi-infarct dementia, Lewy bodydementia, frontal lobe dementia such as Pick's disease, subcorticaldementias (such as Huntington or progressive supranuclear palsy), focalcortical atrophy syndromes (such as primary aphasia), metabolic-toxicdementias (such as chronic hypothyroidism or B12 deficiency), anddementias caused by infections (such as syphilis or chronic meningitis).

Alzheimer's disease is characterized by extracellular deposits ofβ-amyloid protein (β-AP) in senile plaques and cerebral vessels. β-AP isa peptide fragment of 39 to 42 amino acids derived from an amyloidprotein precursor (APP). At least three isoforms of APP are known (695,751, and 770 amino acids). Alternative splicing of mRNA generates theisoforms; normal processing affects a portion of the β-AP sequence,thereby preventing the generation of β-AP. It is believed that abnormalprotein processing by the proteasome contributes to the abundance ofβ-AP in the Alzheimer brain. The APP-processing enzyme in rats containsabout ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has anN-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which isidentical to the β-subunit of human macropain (Kojima, S. et al., Fed.Eur. Biochem. Soc., (1992) 304:57-60). The APP-processing enzyme cleavesat the Gln¹⁵--Lys¹⁶ bond; in the presence of calcium ion, the enzymealso cleaves at the Met-¹--Asp¹ bond, and the Asp¹--Ala² bonds torelease the extracellular domain of β-AP.

One aspect of the invention, therefore, relates to a method of treatingAlzheimer's disease, comprising administering to a subject an effectiveamount of the proteasome inhibitor composition disclosed herein. Suchtreatment includes reducing the rate of β-AP processing, reducing therate of β-AP plaque formation, reducing the rate of β-AP generation, andreducing the clinical signs of Alzheimer's disease.

Fibrosis is the excessive and persistent formation of fibrous connectivetissue resulting from the hyperproliferative growth of fibroblasts andis associated with activation of the TGF-β signaling pathway. Fibrosisinvolves extensive deposition of extracellular matrix and can occurwithin virtually any tissue or across several different tissues.Normally, the level of intracellular signaling protein (Smad) thatactivate transcription of target genes upon TGF-β stimulation isregulated by proteasome activity (Xu et al., 2000). However, accelerateddegradation of the TGF-β signaling components has been observed infibrotic conditions, such as cystic fibrosis, injection fibrosis,endomyocardial fibrosis, idiopathic pulmonary fibrosis, myelofibrosis,retroperitoneal fibrosis, progressive massive fibrosis, nephrogenicsystemic fibrosis. Other conditions that are often associated withfibrosis include cirrhosis, diffuse parenchymal lung disease,post-vasectomy pain syndrome, tuberculsis, sickle-cell anemia andrheumatoid arthritis. An embodiment of the invention is the method oftreating a fibrotic or fibrotic-associated condition comprisingadministering an effective amount of the composition described herein toa subject in need of such treatment.

The treatment of burn victims is often hampered by fibrosis, thus, incertain embodiments, the invention relates to the topical or systemicadministration of the inhibitors to treat burns. Wound closure followingsurgery is often associated with disfiguring scars, which may beprevented by inhibition of fibrosis. Thus, in certain embodiments, theinvention relates to a method for the prevention or reduction ofscarring.

Overproduction of lipopolysaccharide (LPS)-induced cytokines such asTNFα is considered to be central to the processes associated with septicshock. Furthermore, it is generally accepted that the first step in theactivation of cells by LPS is the binding of LPS to specific membranereceptors. The α- and β-subunits of the 20S proteasome complex have beenidentified as LPS-binding proteins, suggesting that the LPS-inducedsignal transduction may be an important therapeutic target in thetreatment or prevention of sepsis (Qureshi, N. et al., J. Immun. (2003)171: 1515-1525). Therefore, in certain embodiments, the proteasomeinhibitor composition may be used for the inhibition of TNFα to preventand/or treat septic shock.

Ischemia and reperfusion injury results in hypoxia, a condition in whichthere is a deficiency of oxygen reaching the tissues of the body. Thiscondition causes increased degradation of Iκ-Bα, thereby resulting inthe activation of NF-κB (Koong et al., 1994). It has been demonstratedthat the severity of injury resulting in hypoxia can be reduced with theadministration of a proteasome inhibitor (Gao et al., 2000; Bao et al.,2001; Pye et al., 2003). Therefore, certain embodiments of the inventionrelate to a method of treating an ischemic condition or reperfusioninjury comprising administering to a subject in need of such treatmentan effective amount of the proteasome inhibitor compound disclosedherein. Examples of such conditions or injuries include, but are notlimited to, acute coronary syndrome (vulnerable plaques), arterialocclusive disease (cardiac, cerebral, peripheral arterial and vascularocclusions), atherosclerosis (coronary sclerosis, coronary arterydisease), infarctions, heart failure, pancreatitis, myocardialhypertrophy, stenosis, and restenosis.

NF-κB also binds specifically to the HIV-enhancer/promoter. Whencompared to the Nef of mac239, the HIV regulatory protein Nef of pbj 14differs by two amino acids in the region which controls protein kinasebinding. It is believed that the protein kinase signals thephosphorylation of IκB, triggering IκB degradation through theubiquitin-proteasome pathway. After degradation, NF-κB is released intothe nucleus, thus enhancing the transcription of HIV (Cohen, J.,Science, (1995) 267:960). In certain embodiments, the invention relatesto a method for inhibiting or reducing HIV infection in a subject, or amethod for decreasing the level of viral gene expression, each methodcomprising administering to the subject an effective amount of theproteasome inhibitor composition disclosed herein.

Viral infections contribute to the pathology of many diseases. Heartconditions such as ongoing myocarditis and dilated cardiomyopathy havebeen linked to the coxsackievirus B3. In a comparative whole-genomemicroarray analyses of infected mouse hearts, specific proteasomesubunits were uniformly up-regulated in hearts of mice which developedchronic myocarditis (Szalay et al, Am J Pathol 168:1542-52, 2006). Someviruses utilize the ubiquitin-proteasome system in the viral entry stepwhere the virus is released from the endosome into the cytosol. Themouse hepatitis virus (MHV) belongs to the Coronaviridae family, whichalso includes the severe acute respiratory syndrome (SARS) coronvirus.Yu and Lai (J Virol 79:644-648, 2005) demonstrated that treatment ofcells infected with MHV with a proteasome inhibitor resulted in adecrease in viral replication, correlating with reduced viral titer ascompared to that of untreated cells. The human hepatitis B virus (HBV),a member of the Hepadnaviridae virus family, likewise requires virallyencoded envelop proteins to propagate. Inhibiting the proteasomedegradation pathway causes a significant reduction in the amount ofsecreted envelope proteins (Simsek et al, J Virol 79:12914-12920, 2005).In addition to HBV, other hepatitis viruses (A, C, D and E) may alsoutilize the ubiquitin-proteasome degradation pathway for secretion,morphogenesis and pathogenesis. Accordingly, in certain embodiments, theinvention relates to a method for treating viral infection, such as SARSor hepatitis A, B, C, D and E, comprising contacting a cell with (oradministering to a subject) an effective amount of the compounddisclosed herein.

In certain embodiments, the disclosed compositions may be useful for thetreatment of a parasitic infection, such as infections caused byprotozoan parasites. The proteasome of these parasites is considered tobe involved primarily in cell differentiation and replication activities(Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore,entamoeba species have been shown to lose encystation capacity whenexposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res.1997, 28, Spec No: 139-140). In certain such embodiments, theadministrative protocols for the proteasome inhibitor compositions areuseful for the treatment of parasitic infections in humans caused by aprotozoan parasite selected from Plasmodium sps. (including P.falciparum, P. vivax, P. malariae, and P. ovale, which cause malaria),Trypanosoma sps. (including T. cruzi, which causes Chagas' disease, andT. brucei which causes African sleeping sickness), Leishmania sps.(including L. amazonesis, L. donovani, L. infantum, L. mexicana, etc.),Pneumocystis carinii (a protozoan known to cause pneumonia in AIDS andother immunosuppressed patients), Toxoplasma gondii, Entamoebahistolytica, Entamoeba invadens, and Giardia lamblia. In certainembodiments, the disclosed proteasome inhibitor compositions are usefulfor the treatment of parasitic infections in animals and livestockcaused by a protozoan parasite selected from Plasmodium hermani,Cryptosporidium sps., Echinococcus granulosus, Eimeria tenella,Sarcocystis neurona, and Neurospora crassa. Other compounds that act asproteasome inhibitors in the treatment of parasitic diseases aredescribed in WO 98/10779, which is incorporated herein in its entirety.

In certain embodiments, the proteasome inhibitor compositions inhibitproteasome activity in a parasite without recovery in red blood cellsand white blood cells. In certain such embodiments, the long half-lifeof blood cells may provide prolonged protection with regard to therapyagainst recurring exposures to parasites. In certain embodiments, theproteasome inhibitor compositions may provide prolonged protection withregard to chemoprophylaxis against future infection.

Prokaryotes have what is equivalent to the eukaryote 20S proteasomeparticle. Albeit, the subunit composition of the prokaryote 20S particleis simpler than that of eukaryotes, it has the ability to hydrolyzepeptide bonds in a similar manner. For example, the nucleophilic attackon the peptide bond occurs through the threonine residue on theN-terminus of the β-subunits. Thus, an embodiment of this inventionrelates to a method of treating prokaryotic infections, comprisingadministering to a subject an effective amount of the proteasomeinhibitor composition disclosed herein. Prokaryotic infections mayinclude diseases caused by either mycobacteria (such as tuberculosis,leprosy or Buruli Ulcer) or archaebacteria.

It has also been demonstrated that inhibitors that bind to the 20Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, thedisclosed proteasome inhibitor composition may be useful in thetreatment and/or prevention of diseases associated with bone loss, suchas osteoporosis.

Thus, in certain embodiments, the invention relates to a method fortreating a disease or condition selected from cancer, autoimmunedisease, graft or transplant-related condition, neurodegenerativedisease, fibrotic-associated condition, ischemic-related conditions,infection (viral, parasitic or prokaryotic) and diseases associated withbone loss, comprising administering a crystalline compound of Formula(II).

Compounds prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and ifdesired, the active ingredient may be mixed with any conventionaladditive or excipient, such as a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions of the present invention arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other cases, the inhibitors useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acomposition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, cyclodextrins, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the composition. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the inhibitor(s) ina polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microcapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compositions of this invention may beprovided in an aqueous solution containing about 0.1-10% w/v of acompound disclosed herein, among other substances, for parenteraladministration. Typical dose ranges are from about 0.01 to about 50mg/kg of body weight per day, given in 1-4 divided doses. Each divideddose may contain the same or different compounds of the invention. Thedosage will be an effective amount depending on several factorsincluding the overall health of a patient, and the formulation and routeof administration of the selected compound(s).

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆alkoxyalkyl” refers to a C₁₋₆alkyl group substituted withan alkoxy group, thereby forming an ether.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, the amino group is basic,meaning the protonated form has a pK_(a)≧7.00.

The terms “amide” and “amido” are art-recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R^(11′) represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above.Where X is an oxygen and R¹¹ or R^(11′) is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R¹¹ is a hydrogen, theformula represents a “carboxylic acid”.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. The term“heteroaryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, forexample, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms. The term terms “heterocyclyl” or “heterocyclic group”also include polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings wherein atleast one of the rings is heterocyclic, e.g., the other cyclic rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example, piperidine,piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term “C₁₋₆hydroxyalkyl” refers to a C₁₋₆alkyl group substituted witha hydroxy group.

The terms “polycyclyl” or “polycyclic” refer to two or more rings (e.g.,cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Each of the rings of thepolycycle can be substituted or unsubstituted.

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “substantially pure” as used herein, refers to a crystallinepolymorph that is greater than 90% pure, meaning that contains less than10% of any other compound, including the corresponding amorphouscompound. Preferably, the crystalline polymorph is greater than 95%pure, or even greater than 98% pure.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “thioether” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In preferred embodiments, the“thioether” is represented by —S-alkyl. Representative thioether groupsinclude methylthio, ethylthio, and the like.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

EXEMPLIFICATION Example 1 Synthesis of Compound 1

Synthesis of (B)

Hydroxybenztriazole (HOBT) (10.81 g, 80.0 mmol) and DIEA (200.0 mmol,25.85 g, 35 mL) was added to a solution of NBoc leucine (50.0 mmol,11.56 g) and phenylalanine methyl ester (50.0 mmol, 10.78 g) in 500 mLof DMF. The mixture was cooled to 0° C. in an ice-water bath andbenzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate(BOP) (80.0 mmol, 35.38 g) was added in several portions over fiveminutes. The reaction was placed under an atmosphere of argon andstirred overnight. The reaction was diluted with brine (1000 mL) andextracted with EtOAc (5×200 mL). The organic layers were combined andwashed with water (10×100 mL) and brine (2×150 mL) and dried over MgSO₄.The MgSO₄ was removed by filtration and the volatiles removed underreduced pressure to give (A) (18.17 g). To a 50 mL 0° C. cooled solutionof 80% TFA/DCM was added BocNHLeuPheOMe (45.86 mmol, 18.0 g). Thesolution was stirred and allowed to warm to room temperature over 2 hr.The volatiles were removed under reduced pressure to give an oil.BocNHhPhe (45.86 mmol, 12.81 g), DMF (500 mL), HOBT (73.37 mmol, 9.91 g)and DIEA (183.44 mmol, 23.70 g, 32.0 mL) were then added to the oil. Themixture was cooled to 0° C. in an ice-water bath and BOP (73.37 mmol,32.45 g) was added in several portions over five minutes. The reactionwas placed under argon and allowed to warm to room temperatureovernight. The reaction was diluted with H₂O (1500 mL) and extractedwith DCM (5×300 mL). The organic layers were combined and washed withH₂O (6×300 mL) and brine (1×300 mL) and dried over MgSO₄. The MgSO₄ wasremoved by filtration and the volatiles removed under reduced pressureto give a yellow solid. EtOH (200 mL, 95%) was then added to the yellowsolid and the mixture was heated to 65° C. to dissolve all of thesolids. The solution was then added to 1000 mL of chilled H₂O and theresulting precipitate collected to give (B) (21.59 g).

Synthesis of (C)

(B) (1.80 mmol, 1.0 g) was mixed with TFA/DCM (80%) and was stirred atroom temperature for 1 hr, at which time the mixture was concentratedand placed under high vacuum for 2 hr giving the TFA salt of thetri-peptide amine. To a 0° C. solution of the TFA salt (1.80 mmol) inDMF (10 mL) was added DIEA (3.6 mmol, 0.7 mL) followed by chloroacetylchloride (2.7 mmol, 0.215 mL). The reaction was allowed to warm to RTwhile stirring overnight under an atmosphere of nitrogen. The mixturewas then diluted with brine (15 mL) and extracted with EtOAc (3×15 mL).The organic layers were combined, washed with H₂O (2×15 mL) and brine(2×15 mL) and dried over Na₂SO₄. The Na₂SO₄ was removed by filtrationand the volatiles removed under reduced pressure. The crude material wassuspended in EtOAc and filtered to give (C) (0.640 g)

Synthesis of (D)

KI (0.019 mmol, 0.0032 g) and morpholine (0.110 mmol, 0.0096 g) wereadded to a solution of (C) (0.094 mmol, 0.050 g) in THF (10 mL) and themixture was stirred overnight under an atmosphere of nitrogen. Thevolatiles were removed under reduced pressure and the crude materialtaken up in EtOAc (15 mL), washed with H₂O (2×10 mL) and brine (2×10 mL)and dried over MgSO₄. The MgSO₄ was removed by filtration and thevolatiles removed under reduced pressure to give (D).

Synthesis of (E)

LiOH (0.94 mmol, 0.023 g) was added to a slurry of (D) (0.094 mmol) in 4mL of 3:1 MeOH/H₂O cooled to 0° C. After 12 hr at 5° C. the reaction wasquenched with 20 mL sat. NH₄Cl and diluted further with 10 mL H₂O. ThepH of the reaction mixture was adjusted to 3 with 1 N HCl, extractedwith DCM (3×15 mL), and dried over MgSO₄. The MgSO₄ was removed byfiltration and the volatiles were removed under reduced pressure to give(E).

Synthesis of Compound 1

(E) (0.082 mmol, 0.046 g), DIEA (0.328 mmol, 0.057 mL) and HOBT (0.133mmol, 0.018 g) were added to a stirred solution of (F) (0.082 mmol) inDMF (2 mL). The mixture was cooled to 0° C. in an ice bath and BOP(0.131 mmol, 0.058 g) was added in several portions. The mixture wasstirred at 5° C. under an atmosphere of argon overnight. The reactionwas then diluted with H₂O (15 mL) and extracted with EtOAc. The organiclayer was washed with water, sat. NaHCO₃, and brine and dried overanhydrous MgSO₄. The MgSO₄ was removed by filtration and the volatilesremoved under reduced pressure to give compound 1 (0.034 g) (IC₅₀ 20SCT-L<100 nM; IC₅₀ Cell-Based CT-L<100 nM).

Example 2

Compound 1 (1.0 g) was dissolved in methanol (16 mL) heated to 80° C.Water (4 mL) was then slowly added and the clear solution was allowed tocool to ambient temperature and the solution was brought tosupersaturation by evaporating off 10 mL of solvent with compressed air.The resulting crystals were filtered, washed with 8 mL 1:1 deionizedwater-methanol, and dried under vacuum for 12 hours to providecrystalline compound 1 (0.9 g) with a melting point of 212° C.

The characteristic DSC curve of the sample is shown in FIG. 1 asrecorded on a TA Instruments Differential Scanning calorimeter 2920 at aheating rate of 10° C./minute.

Example 3

Compound 1 (1.0 g) was dissolved in acetonitrile (17 mL) heated to 80°C. Water (8 mL) was then slowly added and the clear solution was allowedto cool to ambient temperature and the solution was brought tosupersaturation by evaporating off 10 mL of solvent with compressed air.The resulting crystals were filtered, washed with 8 mL 1:1 deionizedwater-acetonitrile, and dried under vacuum for 12 hours to providecrystalline compound 1 (0.85 g) with a melting point of 212° C.

Example 4

Compound 1 (1.0 g) was dissolved in ethanol (17 mL) heated to 80° C.Water (5 mL) was then slowly added and the clear solution was allowed tocool to ambient temperature and the solution was brought tosupersaturation by evaporating off 15 mL of solvent with compressed air.The resulting crystals were filtered, washed with 8 mL 1:1 deionizedwater-ethanol, and dried under vacuum for 12 hours to providecrystalline compound 1 (0.82 g) with a melting point of 212° C.

Example 5

Compound 1 (1.0 g) was dissolved in ethyl acetate (30 mL) heated to 80°C. Water (5 mL) was then slowly added and the clear solution was allowedto cool to ambient temperature and the solution was brought tosupersaturation by evaporating off 20 mL of solvent with compressed air.The resulting crystals were filtered, washed with 5 mL ethyl acetate,and dried under vacuum for 12 hours to provide crystalline compound 1(0.60 g) with a melting point of 212° C.

Example 6

Compound 1 (1.0 g) was dissolved in ethanol (15 mL) heated to 80° C.Water (5 mL) was then slowly added and the clear solution was allowed tocool to ambient temperature and the solution was brought tosupersaturation by evaporating off 10 mL of solvent with compressed air.The resulting crystals were filtered, washed with 10 mL 1:1 deionizedwater-ethanol, and dried under vacuum for 12 hours to providecrystalline compound 1 (0.54 g) with a melting point of 212° C.

Example 7

Synthesis of (F)

Compound (G) (0.43 g) was prepared according to U.S. Pat. ApplicationNo. 2005-0256324 and was added to a flask along with Pd/C (10% wt, 0.10g) followed by slow addition of TFA (35 mL). The flask was evacuated andback-flushed with hydrogen gas three times and then the reaction mixturewas stirred under one atmosphere of hydrogen at room temperature for twohours. The reaction mixture was then filtered through Celite and thefiltrate was concentrated under reduced pressure. Dichloromethane (25mL) was added and the volatiles removed under reduced pressure. Theresultant thick yellow syrup was dried under high vacuum to a constantweight. The syrup was then transferred to 50 mL volumetric flask andrinsed with 8.5 mL diethyl ether to yield crystalline compound (F) (0.33g).

Synthesis of Compound 1

A 10 mL volumetric flask was charged with 1-hydroxybenzotriazole (HOBT,0.54 g) and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU, 1.54 g) and diluted to 50 mL with DMF. Thisstock solution of coupling reagents was 0.40 M for both HOBT and HBTU.

(E) (0.61 g), (F) (0.33 g), and the coupling reagent stock solution (2.7mL), were added to a 10 mL volumetric flask and the mixture was cooledto 0° C. DIEA (0.56 mL) was then added dropwise to the cooled solution.The mixture was allowed to stir at 0° C. for 60 minutes and was thenquenched by the addition of saturated sodium bicarbonate (15 mL). Themixture was diluted with ethyl acetate (35 mL) and the layers separated.The organic layer was washed saturated sodium bicarbonate (3×15 mL),brine (2×15 mL) and dried over sodium sulfate. The sodium sulfate wasremoved by filtration and the volatiles removed under reduced pressureto give a thick syrup which was further dried under high vacuum to givea crude compound 1 as a foam (0.59 g).

Example 8

Crude compound 1 (0.590 g) was completely dissolved in methanol (11 mL)by stirring and heating in an oil bath (80° C.) and deionized water (17mL) was added dropwise. The mixture was seeded with crystalline compound1, stirred and allowed to slowly evaporate for 12 hours to approximately20 mL to precipitate compound 1. The suspension was filtered, washedwith 1:1 deionized water-methanol (4 mL), and dried under vacuum for 12hours at room temperature to yield compound 1 as a white solid (0.25 g).The crystallization was repeated two additional times to yieldcrystalline compound 1 (0.13 g).

Crystalline compound 1 (0.3 g) was dissolved in isopropanol (15 mL) bystirring and heating in an oil bath (80° C.). The solution wasconcentrated under reduced pressure to reduce volume to 5 mL. Deionizedwater (20 mL) was quickly added and the resultant suspension wasrigorously stirred for 1 hour. The glassy precipitate was filtered,rinsed with deionized water (25 mL) and dried to yield amorphousCompound 1 (0.3 g).

The characteristic DSC curve of the amorphous sample is shown in FIG. 7which was recorded on a TA Instruments Differential Scanning calorimeter2920 at a heating rate of 1° C./minute for the amorphous form ofCompound 1.

The characteristic X-ray diffraction pattern of the amorphous powder isshown in FIG. 8 and was recorded on the Shimadzu XRD-6000 under Cu Kαradiation [voltage and current set at 40 kV and 40 mA; divergence andscattering slits set at 1° and receiving slit set at 0.15 mm; NaIscintillation detector used for diffracted radiation; a 0-20 continuousscan at 3°/min (0.4 sec/0.02° step) from 2.5 to 40° 2θ was used; sampleswere placed in an aluminum holder with silicon insert; and datacollected and analyzed with XRD-6100/7000 v.5.0].

Example 9 Synthesis of (F)

A flask was charged with (G) and ethyl acetate (400 mL) and the solutionwas cooled in an ice bath for 15 minutes with stirring. Trifluoroaceticacid (200 mL) was added dropwise, maintaining an internal temperature ofless than 10° C. Pd/C (3.6 g) was added in one portion and the flask waspurged under high vacuum and refilled with hydrogen three times. After 2hours, the reaction was filtered through Celite and the filtrateevaporated under reduced pressure to a thick orange oil which wasswirled gently with 170 mL diethyl ether. As the flask was swirled, finecrystals formed. The flask was allowed to sit at room temperature, andrapid crystallization occurred. After 1 hour at ambient temperature, theflask was capped tightly and placed in the freezer overnight (<−5° C.).The resulting crystalline solid was filtered and washed with ice coldethyl ether (50 mL) and dried under high vacuum. Fine white crystals(14.1 g; melting point: 137° C.) of (F) were obtained.

Synthesis of Compound 1

A flask was charged with (F) (10 g), (E) (15.3 g), HBTU (15.3 g), HOBt(5.5 g), and DMF (300 mL). The mixture was stirred vigorously untildissolved and was placed in an NaCl/ice bath (−5° C.). After 15 minutes,DIEA (7.1 mL) was added dropwise over <10 minutes, maintaining aninternal temperature of less than −3° C. After addition was complete,the reaction mixture was stirred in the bath for one hour and wasquenched by addition of saturated NaHCO₃ (aq.) (200 mL). The slurry wasextracted with ethyl acetate (1.5 L) and the organic layer was washedwith sat. NaHCO₃(aq.) (2×300 mL) and sat. NaCl (aq.) (200 mL), and thendried over MgSO₄.

The organic layer was concentrated to ˜50 mL under reduced pressure andmethylethyl ketone (200 mL) was added, and the solution was againconcentrated to ˜50 mL. Methylethyl ketone (125 mL) was added again, andthe solution was stirred in an oil bath (80° C.) until clear. Thesolution was then allowed to cool and was seeded with pure crystallineCompound 1. The mixture was stirred for 2 hours at 25° C. and thenovernight at 0° C. The white solid precipitate was filtered and washedwith ice cold methylethyl ketone (300 mL) to give white solids. Thesolid was dried under high vacuum at ambient temperature to a constantweight to yield 13.5 g of pure compound 1.

Example 10

Synthesis of (F)

A flask was charged with (H) (100 g) [see: Bioorg. Med. Chem. Letter1999, 9, 2283-88], and dichloromethane (300 mL) under nitrogen and thesolution was cooled in an ice bath to 0-5° C. Trifluoroacetic acid(136.9 mL) was added dropwise with stirring at 0-10° C., after which thereaction mixture was removed from the ice bath and stirred at roomtemperature for 2 hours. Methyl tert-butyl ether (300 mL) was then addedand 400 mL of solvent was evaporated under reduced pressure. MTBE (200mL) was then added via addition funnel and the solution stirred for 20minutes at 20° C., then heptanes (1000 mL) were added within 10 minutesand the reaction mixture cooled to 0-5° C. The reaction mixture wasstirred for 30 minutes and then the solids were filtered, rinsed withcold heptanes (0-5° C., 3×100 mL) and dried under high vacuum to theconstant weight to yield 90.69 g of (F) as a white solid.

Synthesis of Compound 1

A solution of (F) (137.53 g) in DMF (900 mL) was cooled in a NaCl/icebath to −2° C. HBTU (138.06 g), HOBT (55.90 g), (F) (90.00 g) and icecold DMF (180 mL) were then added to the solution followed by additionof neat DIEA (67.19 g, 509.66 mmol) via a dropping funnel at a rate suchthat the internal temperature remained at ˜0° C. After two hours, neatisopropylethylamine (24.0 g) was added via a dropping funnel. Themixture was stirred at 0° C. until conversion >99%. The reaction mixturewas then transferred portionwise into a dropping funnel and slowly addedto an ice cold half-saturated NaHCO₃ solution (3.6 L) (internaltemperature maintained at 20° C.). The resulting slurry was stirred witha mechanical stirrer for 30 minutes and the solids were then filteredand the filter cake washed with ice cold water (2×1350 mL). The solidswere then dissolved in dichloromethane (2.7 L) and the organic phase wasextracted with water (portions of 2700 mL) until relative percent areafor HOBt/HBTU was <15% by HPLC (200 μL solution for HPLC sample). Theorganic phase was filtered through a plug of sodium sulfate andsubsequently inline filtered through a pad of active charcoal.

The organic phase was concentrated under reduced pressure andmethylethyl ketone (1350 mL) was added and the solution concentratedagain under reduced pressure. Methylethyl ketone (1350 mL) was thenadded and the solution concentrated again under reduced pressure. Theresulting concentrated solution was cooled to 0° C. until solids wereformed; then the mixture was heated to 75° C. as more methylethyl ketonewas added (ca. 750 mL) until complete dissolution. The solution wascooled to 65° C. and seeded and the resulting solution/slurry was cooledat a rate of 0.5° C./minute to 20° C. (stir rate of 60-70 rpm). Theslurry was stirred for a minimum of 5 hours at 20° C. to allow forcomplete crystallization. The solids were filtered off and washed withice cold methylethyl ketone (720 mL) and the filter cake was dried undera stream of nitrogen for 1 hour. The solids were transferred into around bottom flask and dried under reduced pressure to constant weightto yield 116 g of crystalline compound 1.

Example 11

Methanol (200 mL) was added to crude Compound 1 and the mixture wasconcentrated to 100 mL. Additional methanol (275 mL) was added, alongwith deionized water (75 mL), and the mixture concentrated to 400 mL.The clear solution was then seeded with pure crystalline Compound 1,stirred and allowed to slowly evaporate under a stream of compressed airto 200 mL. The resulting yellowish solid was washed with deionized water(400 mL) and 1:1 deionized water-methanol (300 mL) until it turned whiteand filtrate turned clear. Compound 1 was then dried under vacuum for 12hours.

The resulting compound 1 (17.3 g) was completely dissolved in methanol(275 mL) by stirring and heating in oil bath (bath set at 85° C.;mixture temperature less than 65° C.). Deionized water (75 mL) was addeddropwise over 15 minutes, and the clear mixture was allowed to cool toroom temperature. Seed crystals of compound 1 were added to the stirredsolution, and the mixture was allowed to slowly concentrate under astream of compressed air to approximately 250 mL over 9 hours. Thecrystals were then filtered and washed with 1:1 deionized water-methanol(300 mL). The white solid was dried under vacuum for 12 hours at 22° C.to yield crystalline compound 1 (14.0 g).

Example 12

Crude compound 1 (12.1 g) was completely dissolved in methanol (50 mL)by stirring and heating in oil bath (bath set at 85° C.; mixturetemperature less than 65° C.). The clear solution was allowed to cool toroom temperature and seed crystals of compound 1 were added to thesolution. The mixture was allowed to crystallize over three hours atroom temperature. The resulting solid was washed with 1:1 deionizedwater-methanol (500 mL), filtered, and dried under vacuum for 12 hoursto yield crystalline compound 1 (9.4 g).

Example 13 Synthesis of (F)

A flask was charged with (H) (1 g) and ethyl acetate (20 mL) and thesolution was cooled in an ice bath for 15 minutes with stirring.Trifluoroacetic acid (10 mL) was then added dropwise, while maintainingan internal temperature of less than 3° C. After stirring at 0° C. for 2hours, the reaction was allowed to warm to ambient temperature and wasstirred for two additional hours. The solution was then evaporated underreduced pressure to a thick colorless oil. This crude mixture wasswirled gently with 10 mL of diethyl ether and as the solution wasswirled, fine crystals formed. After 30 minutes at ambient temperature,the flask was capped tightly and placed in the freezer overnight. Theresulting crystalline solid was filtered and washed with ice colddiethyl ether, and then dried on high vacuum to a constant weight togive fine white crystals of (F) (670 mg).

Example 14 Synthesis of Compound 1

Compound (E) (14.2 g), HBTU (14.3 g), HOBT (5.1 g) and DMF (300 mL),were added to (F) and the mixture was stirred at room temperature tocomplete dissolution. The reaction was cooled in ice bath for 15minutes, and DIEA (32 mL) was added over 15 minutes while maintaining aninternal temperature of less than 10° C. The reaction mixture was thenstirred at 0° C. for one hour before it was quenched with saturatedsodium bicarbonate (200 mL). The mixture was extracted with ethylacetate (1.5 L), and the organic layer was washed with saturated sodiumbicarbonate (2×300 mL) and deionized water (1×200 mL). The combinedaqueous wash was extracted with ethyl acetate (200 mL) and the organiclayers were combined (1.7 L).

The combined organic layers (1.7 L) were concentrated under reducedpressure to 100 mL followed by addition of methanol (200 mL), and themixture was again concentrated to 100 mL. Additional methanol (200 mL)was added, deionized water (75 mL) was slowly added with stirring, andthe mixture concentrated to 300 mL. The clear solution was seeded withcrystalline compound 1, stirred and allowed to slowly concentrate undera stream of compressed air to about 200 mL. The off-white solid waswashed until solid turned white and filtrate turned clear with a 4:1deionized water-methanol (2 L) and 1:1 deionized water-methanol (500mL). The resulting solid was dried under vacuum for 12 hours at 22° C.to provide compound 1 (16.8 g).

Compound 1 was completely dissolved in ethanol (200 mL) by stirring andheating in oil bath (bath set at 85° C.; mixture temperature less than65° C.). The clear solution was allowed to cool to room temperature andseed crystals of compound 1 were added to the stirred solution, and themixture was flushed with air and allowed to crystallize. The mixture wasthen filtered, washed with 1:1 deionized water-ethanol (200 mL), anddried under vacuum for 12 hours at room temperature to yield 10.2 g ofcrystalline compound 1.

Example 15 Synthesis of (F)

A 500 mL flask was equipped with a mechanical stirrer, thermocouple,cooling bath. (G) (12.5 g) was dissolved in ethyl acetate (125 mL) andthe clear solution was cooled to 0-5° C. followed by slow addition oftrifluoroacetic acid (375 mL) such that the internal temperature wasmaintained below 10° C. After warming to room temperature, 5% Pd/C (1.25g) was added and the reaction mixture under an atmosphere of hydrogenfor 2 hours. The reaction mixture was filtered through a glass fiber andrinsed with ethyl acetate (50 mL). The filtrate was then concentratedunder reduced pressure to yield a yellow oil. MTBE (50 mL) was added tothe oil and co-evaporated to yellow oil at 25° C. MTBE (60 mL) was againadded and the mixture was cooled to −10° C. and stirred for 60 minutes.Heptanes (120 mL) were then slowly added to the stirred mixture andstirring was continued at −10° C. for an additional 15 minutes. Thesolids were collected by filtration and the crystals were rinsed withheptanes (2×40 mL) and dried under high vacuum at room temperature (22°C.) to a constant weight (10.1 g).

Synthesis of Compound 1

A flask equipped with a mechanical stirrer, thermocouple, cooling bath,nitrogen inlet and drying tube was charged with DMF, (F) (133.9 g), (E)(241.8 g), HBTU (242.8 g), and HOBT (86.5 g) and the mixture was stirredand cooled to 0-5° C. DIEA (156 mL) was then added slowly over at least30 minutes, while maintaining temperature between 0-5° C. The reactionmixture was stirred at 0-5° C. for one hour and was then poured into avigorously stirred saturated solution of sodium bicarbonate (3630 mL)and ethyl acetate (900 mL). Additional ethyl acetate (2000 mL) was addedto extract the product and the organic layer was separated. The aqueouslayer was then extracted with ethyl acetate (1930 mL). The organicphases were combined and washed with saturated solution of sodiumbicarbonate (2420 mL) and brine (2420 mL), dried over magnesium sulfate(360 g), filtered through glass fiber filter and rinsed with ethylacetate (2×360 mL).

The resulting solution was concentrated to a semisolid under reducedpressure and methanol (725 mL) was added and co-evaporated under reducedpressure to yield semi-solid compound 1. The crude product was dissolvedin methanol (5320 mL) and the solution was stirred while water (2130 mL)was added over twenty minutes. When addition of water was complete,approximately 0.3 g of pure crystalline seeds were added and themethanol/water solution was stirred for three hours. The resultingcrystalline white solid was isolated by filtration and the fine whitecrystalline product was rinsed with a methanol/water solution (1:1, 1200mL). The resulting solid was rinsed with methanol/water solution (1:1,1200 mL) and the crystalline product was poured onto drying tray anddried to a constant weight under high vacuum at 27° C. under nitrogenbleed to yield crystalline compound 1 (230 g).

Example 16 Synthesis of (F)

A 100 mL three-neck round bottom flask was charged with (G) (5 g) anddichloromethane (15 mL). The mixture was stirred until the solids haddissolved, and then placed in an ice bath. After 20 minutes, theinternal temperature had reached 0.6° C. and trifluoroacetic acid wasadded dropwise over 5 min. After the addition was complete, the flaskwas allowed to warm to room temperature. After 2 hours, MTBE was addedto the flask (35 mL) and the mixture was cooled in an ice bath, wherein(F) began to crystallize during cooling. Heptanes (65 mL) were thenadded to the flask dropwise over 15 min and the flask was placed in thefreezer (−5° C.). After 1 hour, the solid white product was collectedand washed with heptanes (10 mL) to provide 4.57 g of (F).

Example 17 Synthesis of Compound 1 Citrate Salt

Compound 1 (10 g) and citric acid (2.7 g) were dissolved in THF (75 mL)and acetonitrile (50 mL). The solution was then stirred for 2 hours atroom temperature, at which time a white precipitate formed. The flaskwas then cooled to −10° C. and stirred overnight. The solids werefiltered and washed with 100 mL acetonitrile to give 11.52 g of thecitrate salt of compound 1.

Example 18 Synthesis of (H) and (Q)

Synthesis of (I)

A suspension of dimethyl hydroxylamine hydrochloride (10.53 g, 108 mmol)in DCM (270 mL) under an atmosphere of argon was stirred vigorously for0.5 hours followed by addition of TEA (10.92 g, 14.75 mL, 108 mmol) viaaddition funnel. A solution of Boc-Leucine-OH (25.0 g, 108 mmol) in DCM(270 mL) was cooled to 0° C. followed by dropwise addition ofisobutylchloroformate (14.73 g, 13.98 mL, 108 mmol) via addition funnel.The mixture was further cooled to −20° C. and NMM (10.92 g, 11.87 mL,108 mmol) was added via addition funnel at such a rate to maintain theinternal temperature below −10° C. After stirring for 5 minutes at −20°C., the previously prepared dimethylhydroxylamine solution was added viaa wide bore Teflon cannula. The reaction mixture was removed from thecooling bath and allowed to warm to room temperature overnight. Themixture was then diluted with water (100 mL) and stirred for 15 minutes.The layers were separated and the aqueous layer extracted with DCM (2×50mL). The organic layers were combined, washed with 1 N HCl (4×150 mL),water (1×150 mL), sat. NaHCO₃ (2×100 mL), brine (1×250 mL) and driedover Na₂SO₄. The Na₂SO₄ was removed by filtration and the volatilesremoved under reduced pressure to give (I) (28.05 g, 102 mmol).

Synthesis of (J)

To a 0° C. solution of (I) (10.0 g, 36.4 mmol,) in 100 mL of dry THF,under an atmosphere of argon was added isopropenyl magnesium bromide(364 mL, 182 mmol, 5.0 eq, 0.5 M solution in THF) dropwise using anaddition funnel. The rate of addition was adjusted such that theinternal reaction temperature was maintained below 5° C. After six hoursthe reaction mixture was poured into 250 mL of sat. NH₄Cl and 500 mL wetice. After stirring for 30 minutes the mixture became clear and thevolatiles were removed under reduced pressure and the crude materialdiluted with EtOAc (200 mL). The layers were separated and the aqueouslayer extracted with EtOAc (3×150 mL), the organic layers were combined,washed with water (2×150 mL), brine (2×150 mL) and dried over MgSO₄. TheMgSO₄ was removed by filtration and the volatiles removed under reducedpressure. Purification by flash chromatography (15:1 hexanes/EtOAc) gave(J) as a solid (7.5 g, 29.37 mmol).

Synthesis of (K) and (L)

To a 0° C. solution of (J) (5.0 g, 19.58 mmol) in 200 mL of MeOH wasadded CeCl₃-7H₂O (8.75 g, 23.50 mmol). The solution was stirred under anatmosphere of argon until the CeCl₃-7H₂O was completely dissolved. Tothis solution was added NaBH₄ (0.88 g, 23.50 mmol) in 10 portions over 2minutes. The reaction was then stirred under an atmosphere of argon at0° C. for 6 hours. The reaction was quenched at 0° C. with approximately2.5 mL of glacial HOAc and after 30 minutes of additional stirring at 0°C. the mixture become clear. The volatiles were removed under reducedpressure and the remaining oil taken up in EtOAc (200 mL). The organiclayer was washed with water (2×100 mL), brine (2×100 mL) and dried overMgSO₄. The MgSO₄ was removed by filtration and the volatiles removedunder reduced pressure giving (K) and (L) as a waxy, white solid (4.75g, 18.5 mmol). Ratio of diastereomers 4.5:1 as determined by HPLC.

Synthesis of (M), (N), (O) and (P)

To a solution of (K) and (L) (0.025 g, 0.097 mmol) in DCM (1 mL) wasadded mCPBA (0.018 g, 0.107 mmol). The mixture was stirred at roomtemperature for one hour at which time the mixture was diluted with sat.NaHCO₃ (5 mL). The layers were separated and the aqueous layer extractedwith DCM (2×2 mL). The organic layers were combined and washed withwater (2×5 mL), brine (2×5 mL) and dried over MgSO₄. The MgSO₄ wasremoved by filtration and the volatiles removed under reduced pressureto give an oil.

Synthesis of (H) and (O)

To a solution of Dess-Martin Periodinane (0.023 g, 0.055 mmol) in 1 mLMeCN at 5° C. was added a mixture of (M), (N), (O), and (P) (0.010 g,0.037 mmol) as a solution in MeCN (1 mL). The mixture was placed underan atmosphere of argon and allowed to warm to room temperature whilestirring overnight. When complete, a white precipitate had formed andthe reaction was cooled in an ice-bath and diluted with 2 mL sat.NaHCO₃. The mixture was diluted with 10 mL of EtOAc and the solidsremoved by filtering through a plug of Celite. The mixture wastransferred to a reparatory funnel and the layers separated. The aqueouslayer was extracted with EtOAc (2×5 mL) and the organic layers combined,washed with water (3×5 mL) and brine (1×10 mL) and then dried overNa₂SO₄. The Na₂SO₄ was removed by filtration and the volatiles removedunder reduced pressure to give a mixture of (H) and (O) as a light,yellow oil.

Example 19 Alternate Synthesis of (H) and (Q)

Alternate Synthesis of (H) and (Q)

To a −5° C. solution of (R) (0.200 g, 0.78 mmol) in pyridine (3 mL) wasadded 10% aqueous NaOCl (1.5 mL) dropwise at a rate such that theinternal reaction temperature remained below −4° C. After the additionof NaOCl was complete, the reaction flask was placed in a 0° C. bath andstirred for two hours. The mixture was then diluted with EtOAc (10 mL),washed with water (2×10 mL), brine (2×10 mL) and dried over Na₂SO₄. TheNa₂SO₄ was removed by filtration and the volatiles removed under reducedpressure to give the crude mixture of (H) and (Q). Purification by flashchromatography (20:1 hexanes/EtOAc) gave (H) as an oil (0.059 g, 0.216mmol) and (Q) as a solid (0.023 g, 0.085 mmol).

Example 20 Synthesis of Compound 1

Synthesis of (F)

To a 10 mL round bottomed flask was added (H) (0.050 g, 0.18 mmol) andDCM (0.80 mL). The mixture was cooled to 0° C. and neat TFA (0.20 mL)was added dropwise. After the addition of TFA was complete the flask wasallowed to warm to room temperature while stirring for one hour. Thevolatiles were then removed under reduced pressure and the resulting oilwas chased with DCM (2 mL×2) and the volatiles removed under reducedpressure.

Synthesis of Compound 1

To a 10 mL round bottomed flask containing (F) was added (E) (0.085 g,0.15 mmol), MeCN (2.0 mL), HOBT (0.031 g, 0.23 mmol), and HBTU (0.087 g,0.23 mmol) and the mixture was cooled to 0° C. To this mixture wasslowly added DIEA (0.077 g, 0.104 mL, 0.6 mmol) and the mixture wasallowed to stir at 0° C. for one hour before quenching with saturatedNaHCO₃ (5 mL). The mixture was diluted with EtOAc (15 mL) and the layerswere separated. The organic layer was washed with saturated NaHCO₃ (3×5mL), brine (2×5 mL) and dried over Na₂SO₄. The Na₂SO₄ was removed byfiltration and the volatiles removed under reduced pressure to give athick oil. To the flask containing the oil was added DCM (1 mL) and theplaced under high vacuum while swirling giving Compound 1 (0.100 g, 0.14mmol) as a foam.

Example 21 Synthesis of Compound 1

Alternate Synthesis of (S)

To a 10 mL round bottomed flask was added (G) (0.055 g, 0.18 mmol),formic acid (2 mL), and Pd/C (5% wt, 0.05 g). Once the deprotection wasdeemed complete by TLC and LCMS, the volatiles were removed underreduced pressure. The oil was chased with DCM (2 mL×2) and the volatilesremoved under reduced pressure.

Synthesis of Compound 1

To a 10 mL round bottomed flask containing (S) was added (E) (0.085 g,0.15 mmol), MeCN (2.0 mL), HOBT (0.031 g, 0.23 mmol), HBTU (0.087 g,0.23 mmol) and the mixture was cooled to 0° C. To this mixture wasslowly added DIEA (0.077 g, 0.104 mL, 0.6 mmol). The mixture was thenallowed to stir at 0° C. for 60 minutes and was quenched by the additionof saturated NaHCO₃ (5 mL). The mixture was diluted with EtOAc (15 mL)and the layers separated. The organic layer was washed with saturatedNaHCO₃ (3×5 mL), brine (2×5 mL) and dried over Na₂SO₄. The Na₂SO₄ wasremoved by filtration and the volatiles removed under reduced pressureto give a thick oil. To the flask containing the oil was added DCM (1mL) and the mixture placed under high vacuum while swirling givingCompound 1 as a foam.

Example 22 Synthesis of (H)

Water (214 mL) was added to a three neck flask equipped with amechanical stirrer, an addition funnel, and a thermocouple with displayand cooled to an internal temperature of −5 to 0° C. Solid calciumhypochlorite (107 g, 748 mmol) was then added over approximately 5minutes, while the temperature of the mixture is maintained atapproximately −5° C. to 0° C. The mixture was then further cooled to−10° C. to −5° C. and stirred for 10 minutes followed by addition of NMP(1000 mL) via addition funnel at a rate to maintain internal temperaturebetween −10° C. to −5° C. The reaction slurry was then stirred at −10°C. for 15 minutes. (R) (47.8 g, 187 mmol) was dissolved in NMP (40 0 mL)and added dropwise to the reaction mixture while maintaining theinternal temperature between −15° C. and −10° C. The reaction mixturewas then stirred at −5° C. to 0° C. until the reaction was complete byTLC. Upon reaction completion, the mixture was quenched by slow additionof 1.0 M sodium thiosulfate solution (500 mL), maintaining an internaltemperature of −10° C. to −5° C. Ethyl Acetate (1000 mL) was then added,the layers were separated and the aqueous layer was extracted twicemore. The combined organic layers were washed with water (500 mL) andbrine (500 mL), dried over magnesium sulfate, filtered and concentratedunder reduced pressure to a to yellow oil which was dissolved in hexanes(600 mL) and filtered through a plug of silica to provide (H) as a paleyellow oil (20.8 g).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

1.-18. (canceled)
 19. A method for preparing a crystalline salt of acompound of Formula (II)

wherein the salt is selected from a citrate, tartrate, trifluoroacetate,methanesulfonate, toluenesulfonate, chloride, and bromide salt; and themethod comprises (i) preparing a solution of a compound of Formula (II)in an organic solvent; (ii) adding an acid selected from citric,tartaric, trifluoroacetic, methanesulfonic, toluenesulfonic,hydrochloric, and hydrobromic; (iii) bringing the solution tosupersaturation to cause formation of crystals; and (iv) isolating thecrystals.
 20. A method of claim 19, wherein the organic solvent isselected from diethyl ether, THF, acetonitrile, and MTBE, or anycombination thereof.
 21. A method of claim 20, wherein the organicsolvent is a mixture of THF and acetonitrile.
 22. A method of claim 19,wherein bringing the solution to supersaturation comprises slow additionof an anti-solvent, allowing the solution to cool, reducing the volumeof the solution, or any combination thereof.
 23. A method of claim 22,wherein bringing the solution to supersaturation comprises cooling thesolution to ambient temperature or lower.
 24. A method of claim 19,further comprising washing the crystals.
 25. A method of claim 24,wherein the washing comprises washing with a liquid selected fromdiethyl ether, THF, acetonitrile, and MTBE, or any combination thereof.26. A method of claim 25, wherein washing comprises washing withacetonitrile.
 27. A method of claim 19, wherein isolating the crystalscomprises filtering the crystals.
 28. A method of claim 19, furthercomprising drying the crystals under reduced pressure.
 29. A crystallinesalt of a compound having a structure of Formula (II)

wherein the salt is a citrate salt.
 30. A crystalline salt of claim 29,having a DSC thermogram substantially as shown in FIG.
 11. 31. Acrystalline salt of claim 29, having a melting point of about 180 toabout 190° C.
 32. A crystalline salt of claim 31, having a melting pointof about 184 to about 188° C.
 33. A crystalline salt of claim 29, havingan XRPD pattern substantially as shown in FIG.
 12. 34. A crystallinesalt of claim 29, having 2θ values 4.40; 7.22; 9.12; 12.36; 13.35;14.34; 15.54; 16.14; 16.54; 17.00; 18.24; 18.58; 19.70; 19.90; 20.30;20.42; 21.84; 22.02; 23.34; 23.84; 24.04; 24.08; 24.48; 24.76; 25.48;26.18; 28.14; 28.20; 28.64; 29.64; 31.04; 31.84; 33.00; 33.20; 34.06;34.30; 34.50; 35.18; 37.48; 37.90; 39.48.
 35. A method for thepreparation of a crystalline compound of Formula (III),

wherein X is any suitable counterion, comprising (i) preparing asolution of a compound of Formula (IV) in an organic solvent, wherein PGis a suitable protecting group

(ii) adding a suitable acid; (iii) bringing the solution tosupersaturation to cause formation of crystals; and (iv) isolating thecrystals.
 36. A method of claim 35, wherein PG is selected from Boc andCbz and X is trifluoroacetate.
 37. A method of claim 35, wherein theorganic solvent is selected from dichloromethane, ethyl acetate,isopropyl acetate, isobutyl acetate, butyl acetate, propyl acetate,diethyl ether, methyl tert-butyl ether (MTBE), or any combinationthereof.
 38. A method of claim 37, wherein the organic solvent isselected from dichloromethane, ethyl acetate, MTBE, or any combinationthereof.
 39. A method of claim 35, wherein bringing the solution tosupersaturation comprises addition of an anti-solvent, allowing thesolution to cool, reducing the volume of the solution, or anycombination thereof.
 40. A method of claim 39, wherein bringing thesolution to supersaturation comprises adding an anti-solvent, coolingthe solution to ambient temperature or lower, and reducing the volume ofthe solution.
 41. A method of claim 39, wherein the anti-solvent ishexanes or heptanes.
 42. A method of claim 35, further comprisingwashing the crystals.
 43. A method of claim 42, wherein the washingcomprises washing with a liquid selected from anti-solvent,dichloromethane, ethyl acetate, isopropyl acetate, isobutyl acetate,butyl acetate, propyl acetate, diethyl ether, and methyl tert-butylether, or any combination thereof.
 44. A method of claim 43, whereinwashing comprises washing with anti-solvent.
 45. A method of claim 44,wherein the anti-solvent is hexanes or heptanes.
 46. A method of claim35, wherein isolating the crystals comprises filtering the crystals. 47.A method of claim 35, further comprising drying the crystals underreduced pressure.
 48. A crystalline compound having a structure ofFormula (III)

wherein X is trifluoroacetate.
 49. A crystalline compound of claim 48,having a DSC thermogram substantially as shown in FIG.
 9. 50. Acrystalline compound of claim 48, having an XRPD pattern substantiallyas shown in FIG.
 10. 51. A crystalline compound of claim 48, having 2θvalues 8.84; 15.18; 15.32; 16.20; 16.82; 17.66; 18.26; 19.10; 21.20;22.58; 23.06; 23.52; 25.32; 26.58; 28.60; 30.08; 30.48; 30.84; 32.20;36.14; 37.12.
 52. A method for the preparation of a crystalline compoundof Formula (II),

comprising (i) preparing a solution of a compound of Formula (IV)wherein PG is a suitable protecting group, in a first organic solvent

(ii) adding a suitable acid; (iii) bringing the solution tosupersaturation to cause formation of crystals; and (iv) isolating thecrystals to provide a crystalline compound of Formula (III); (v)reacting a crystalline compound of Formula (III)

wherein X is any suitable counterion, with a compound of Formula (V) ina second organic solvent

(vi) preparing a solution of a compound of Formula (II) in the secondorganic solvent; (vii) bringing the solution to supersaturation to causeformation of crystals; and (viii) isolating the crystals to provide acrystalline compound of Formula (II).
 53. A method for the synthesis ofamino acid keto-epoxides according to Scheme (I)

wherein R¹ is selected from a protecting group or a further chain ofamino acids, which itself may be optionally substituted; R² is selectedfrom hydrogen and C₁₋₆alkyl; or R³ is selected from hydrogen, C₁₋₆alkyl,C₁₋₆alkoxyalkyl, heterocyclyl, aryl, heteroaryl, C₁₋₆heteroaralkyl, andC₁₋₆aralkyl; and wherein the method comprises a stereoselectiveepoxidation with an aqueous sodium hypochlorite or calcium hypochloritesolution in the presence of a cosolvent selected from pyridine,acetonitrile, DMF, DMSO, NMP, DMA, THF, and nitromethane.
 54. A methodof claim 53, further comprising removing the protecting group ifnecessary and coupling with a chain of amino acids.
 55. A method fortreating a disease or condition selected from cancer, autoimmunedisease, graft or transplant-related condition, neurodegenerativedisease, fibrotic-associated condition, ischemic-related conditions,infection (viral, parasitic or prokaryotic) and diseases associated withbone loss, comprising administering a crystalline salt of claim 29.